DE60217081T2 - GENES PARTICIPATED IN V (D) J RECOMBINATION AND / OR DNA REPAIR - Google Patents

Abstract

The invention relates to a new gene and protein involved in V(D)J recombination and repair. The invention also relates to methods of diagnosis and therapy using these genes and protein. The invention also relates to transgenic animals over- and under-expressing said gene.

Description

AREA OF INVENTION

The The present invention relates to a novel DNA sequence which is attached to the V (D) J recombination is involved in lymphocytes and their mutation combined heavy Immunodeficiency (SCID) caused. The invention also relates to diagnostic methods, Therapy method and method for screening new compounds, which use this sequence, as well as non-human transgenic ones Animals.

BACKGROUND THE INVENTION

B- and T lymphocytes recognize foreign antigen by specialized Receptors: the immunoglobulins or the T cell receptor (TCR). The are highly polymorphic antigen-recognizing regions of these receptors from variable (V), diversity (D) and joining (J) gene segments composed before being expressed by a mechanism as V (D) J recombination is known to undergo a somatic rearrangement (Tonegawa, 1983). Each V, D and J segment is from recombination signal sequences Flanked (RSSs) consisting of conserved heptamers and nonamers composed by arbitrary sequences with either 12 or 23 nucleotides are separated. RSSs serve as recognition sequences for the V (D) J recombinase.

The V (D) J recombination can be roughly divided into three steps. Initiate the RAG1 and RAG2 protein the relocation process through the detection of the RSS and the introduction of a DNA double strand break (DSB) at the border of the heptamer (Schatz et al., 1989; Oettinger, 1990). RAG1 and RAG2 are the only ones Two factors that are needed to work in a reaction reminiscent of retroviral integration and transposition (van Gent et al., 1996; Roth and Craig, 1998), the DNA cleavage in cell-free Catalyze systems (McBlane et al., 1995, Van Gent et al. 1995; Eastman et al., 1996). It has been shown that three acid residues, DDE Compound Active Body Worn by RAG1 (Kim et al., 1999; Landree et al., 1999; Fugmann et al., 2000). The limited to immature B and T lymphocytes expression of both RAG1 as well as the RAG2 gene limits V (D) J recombination the lymphoid line. At the end of this phase, which is a DNA damage causes the chromosomal DNA with two hairpin-sealed Coding ends (CE) left behind, while the RSSs and the intervening DNA sequences from the chromosomes as blunt phosphorylated signal ends (SE) cut out of the chromosome (Roth et al., 1992, Schlissel et al., 1993; Zhu and Roth, 1995). The subsequent step is the detection and signaling of DNA damage to the DNA repair machinery. From now on are ubiquitous enzymatic activities involved.

The Description of the murine SCID situation caused by a deficiency is characterized by circulating mature B and T lymphocytes (Bosma et al., 1983), as a general DNA repair defect derived from an elevated one Sensitivity to ionizing radiation or other means which cause a DNA DSB provided the link between V (D) J recombination and DNA DSB repair (Fulop, 1990; Biedermann, 1991; Hendrickson, 1991). This was further analyzed by Chinese ovarian cell lines (CHO) confirmed the initial were selected on the basis of their defect in DNA repair and was found to affect one in vitro V (D) J recombination (Taccioli et al., 1993). This led to the description of the Ku70 / Ku80 / DNA-PKcs complex as DNA damage sensor (overview in (Jackson and Jeggo, 1995)). In short, DNA-PKcs is a DNA-dependent protein kinase, which belongs to the phosphoinositol (PI) kinase family, which occurs at the site of the DNA lesion Recruiting interaction with the regulatory complex Ku70 / 80, which binds to DNA ends (Gottlieb and Jackson, 1993). cell from SCID mice lacks DNA-PK activity due a mutation in the gene encoding DNA-PKcs (Blunt et al., 1996; Danska et al., 1996). This severely affects the V (D) J recombination process, which ultimately leads to a halt in both B and T cell development.

More recently, two proteins, NBS1 and γ-H2AX, have been identified at the site of chromosomal rearrangement in the TCR-α locus in thymocytes (Chen et al., 2000). NBS1, mutated in the Nijmegen fracture syndrome, participates in the formation of the RAD50 / MRE11 / NBS1 complex involved in DNA repair (Carney et al., 1998, Varon et al., 1998). γ-H2AX represents the phosphorylated form of histone H2A in response to external damage and is considered to be an important sensor of DNA damage (Rogakou et al., 1998, Rogakou et al., 1999, Paull et al., 2000). The biological implication of this observation is not yet fully understood, but it demonstrates that the RAD50 / MRE11 / NBS1 complex is involved in the detection and signaling of the RAG1 / 2-mediated DNA DSB to the cellular DNA repair machinery with the DNA PK complex can cooperate. In the final phase of the V (D) J rearrangement, the DNA repair Ma As such, the religation of both chromosomal fractured ends is safe. This last step is similar to the well-known non-homologous termini (NHEJ) DNA pathway in the yeast Saccharomyces cerevisiae (reviewed in (Haber, 2000)) using XRCC4 (Li et al., 1995) and DNA ligase IV (Robins and Lindahl, 1996) factor. The recently obtained crystal structure of XRCC4 demonstrates the dumbbell-like conformation of this protein and provides a structural basis for its binding to DNA as well as its association with DNA ligase IV (Junop et al., 2000). All animal models carrying a defective gene from either of the known V (D) J recombination factors, either natural (murine and equine SCID) or generated by homologous recombination, have a profound defect in the lymphoid development program due to arrest of the B cells. and T-cell maturation in early stages (Mombaerts et al., 1992; Shinkai et al., 1992; Nussenzweig et al., 1996; Zhu et al., 1996; Jhappan et al., 1997; Shin et al., 1997) Barnes et al., 1998; Frank et al., 1998; Gao et al., 1998; Gao et al., 1998; Taccioli et al., 1998). In the cases of DNA ligase IV and XRCC4, this phenotype is also accompanied by early embryonic lethality, which is caused by a massive apoptotic death of postmitotic neurons (Barnes et al., 1998, Frank et al., 1998, Gao et al , 1998).

at People are multiple immune deficiency states by a faulty one T and / or B cell development program (Fischer et al., 1997). In about 20% of cases will be a severe combined immunodeficiency (SCID) phenotype through the full Absence of both circulating B and T lymphocytes which is also associated with a defect in the V (D) J recombination process, while Natural Killer (NK) cells are present. Mutations in either RAG1 or in the RAG2 gene make up a subclass of patients with this condition from (Schwarz et al., 1996; Corneo et al., 2000; Villa et al., 2001). In some patients (RS-SCID), the T-B-SCID defect does not become a RAG1 or RAG2 mutation caused and is characterized by increased sensitivity ionizing radiation of both bone marrow cells (CFU-GMs) as well as primary Skin fibroblasts (Cavazzana-Calvo et al., 1993) and a defect in V (D) J recombination in fibroblasts (Nicolas et al., 1998) accompanied.

Even though this condition suggests that RS-SCID could have a common DNA repair defect, the reminiscent of the murine SCID situation, it was found that the DNA-PK activity is normal in these patients, and the involvement of the DNA-PKcs gene was clearly related by genetic means in several blood relatives Families excluded (Nicolas et al., 1996). A role of all other known genes involved in V (D) J recombination / DNA repair are alike as for responsible for the RS-SCID state (Nicolas et al., 1996). The gene that is defective in RS-SCID therefore encodes one not yet described factor. The inventors have recently the locus associated with the disease the short arm of the human Chromosome 10 is mapped in a 6.5cM region by two polymorphic ones Markers D10S1664 and D10S674 (Moshous et al., 2000), a region that has been shown to be of a similar nature SCID state connected is described by Athabascan-speaking American Indians was (A-SCID) (Hu et al., 1988; Li et al., 1998).

SUMMARY THE INVENTION

The The present invention relates to identification and cloning the Artemis gene, located in this region of chromosome 10. Artemis encodes a novel V (D) J recombination and / or DNA repair factor, which is the metallo-β-lactamase superfamily belongs and whose mutations give rise to the human RS-SCID condition.

Especially The present invention relates to an isolated and purified Nucleic acid molecule that is selected from the group consisting of SEQ ID NO: 1, nucleotides 39-2114 SEQ ID NO: 1 or nucleotides 60-2114 of SEQ ID NO: 1.

The The present invention also relates to polypeptides derived from the nucleic acid of the Invention, and various uses with the objects the invention can be made.

DESCRIPTION THE FIGURES

1 Figure 12 is a schematic view of the genomic organization of the Artemis gene, with the exons shown as rectangles, and the various mutations identified in RS-SCID patients.

DESCRIPTION THE PREFERRED EMBODIMENTS

In In a first aspect, the present invention relates to an isolated and purified nucleic acid molecule selected from the group consisting of SEQ ID NO: 1, nucleotides 39-2114 of SEQ ID NO: 1 or nucleotides 60-2114 of SEQ ID NO: 1.

It It is also contemplated that the invention will be the genomic nucleic acid sequence which leads to SEQ ID No. 1 after transcription genomic nucleic acid sequence can easily be selected by a person skilled in the art from SEQ ID No. 1 by means of a screening a library of genomic DNA using a probe which is derived from SEQ ID NO: 1. She can also go out obtained from the sequence with the GenBank accession number AL360083 which is available at the following address: http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=Nucleotide&list_uids = 12584428 & dopt = GenBank.

The The invention also encompasses an isolated and purified nucleic acid molecule which the complement of the isolated nucleic acid molecule of the invention is as above described.

With Nucleic acid, Nucleic sequence, nucleic acid sequence, Polynucleotide, oligonucleotide, polynucleotide sequence, nucleotide sequence, all expressions, used without distinction in the present application be called a specific strand of nucleotides, modified or not, which is a fragment or a region of a nucleic acid defined, the natural or non-natural Nucleotides. It can be a double-stranded DNA, a single-stranded one DNA or transcription products of the DNAs. The sequences according to the invention also include peptide nucleic acid or analogs or sequences with modified nucleotides (phosphorothioates, Methyl phosphonates ...).

With is meant in isolation that the nucleic acid of the invention is not in their natural Chromosome environment is present. The sequences according to the invention are isolated and / or have been cleaned, meaning that they are direct or indirect (e.g., by copy by amplification), wherein their natural environment has been at least partially modified. The nucleic acids that obtained by chemical synthesis are also part of the present invention.

The stringent hybridization conditions can be defined as it in Sambrook et al. ((1989) Molecular cloning: a laboratory manual. 2nd Ed. Cold Spring Harbor Lab., Cold Spring Harbor, New York) with the following conditions: 5 × or 6 × SCC, 50-65 ° C. High stringency Conditions that are also for A hybridization can be used with the following Conditions defined: 6 × SSC, 60-65 ° C.

The DNA-DNA or DNA-RNA hybridization can be performed in two steps: (1) Prehybridization at 42 ° C for 3 h in phosphate buffer (20 mM, pH 7.5) corresponding to 5 or 6 x SSC (1 x SSC) a solution 0.15 M NaCl + 0.015 M sodium citrate), 50% formamide, 7% sodium dodecyl sulfate (SDS), 10 × Denhardt, Containing 5% dextran sulfate and 1% salmon sperm DNA; (2) hybridization for up to 20 h at a temperature of 50-65 ° C, more preferably 60-65 ° C, followed of various washes (about 20 minutes in 2 x SSC + 2% SDS, then 0.1X SSC + 0.1% SDS). The final wash is in 0.2x SSC + 0.1% SDS for about 30 minutes at about 50-65 ° C and / or 0.1X SSC + 0.1% SDS at the same temperature. These high stringency hybridization conditions can be adapted by a professional. In fact, the expert able to vary the conditions of best stringency by varying the concentrations of SSC and SDS and the temperature of hybridization and to determine washing steps.

Of the Expression "Conditions high stringency "refers also on hybridization and washing under conditions which the binding of a nucleic acid molecule, the used for screening, such as an oligonucleotide probe or cDNA molecule probe, allow for highly homologous sequences. An exemplary high stringency wash solution is 0.2x SSC and 0.1% SDS, used at a temperature between 50-65 ° C.

When oligonucleotide probes are used to screen cDNA or genomic libraries, one of the following two high stringency solutions can be used. The first of these is 6x SSC with 0.05% sodium pyrophosphate at a temperature of 35 ° C-62 ° C, depending on the length of the oligonucleotide probe. For example, 14 base pair probes are washed at 35 ° C-40 ° C, 17 base pair probes are washed at 45 ° C-50 ° C, 20 base pair probes are washed at 52 ° C-57 ° C and 23 base pair probes. Probes are washed at 57-63 ° C. The temperature can be increased by 2-3 ° C, when the non-specific background binding seems high. A second high stringency solution uses tetramethylammonium chloride (TMAC) for the washing of oligonucleotide probes. A stringent wash solution is 3M TMAC, 50mM Tris-HCl, pH 8.0 and 0.2% SDS. The wash temperature using this solution is a function of the length of the probe. For example, a 17 base pair probe is washed at about 45-50 ° C.

Two Polynucleotides are referred to as "identical" or "homologous" when the sequence of the nucleotides or amino acid residues in the two sequences the same is when they are for are aligned to a maximum correspondence, as described below. The term "complementary to" is used herein to denote that the complementary sequence with the whole or a specified contiguous Part of a reference polynucleotide sequence is identical. Sequence comparisons between two (or more) polynucleotides or polypeptides are typified by comparing sequences of two optimally aligned sequences over a segment or "comparison window" performed to local regions of sequence similarity to identify and compare. The optimal alignment of Sequences for the comparison can be made by the local homology algorithm of Smith and Waterman, Ad. App. Math 2: 482 (1981), by the homology alignment algorithm Biol. 48: 443 (1970) by Neddleman and Wunsch, J. Mol Method of finding similarity by Pearson and Lipman, Prop. Natl. Acad. Sci. (U.S.A.) 85: 2444 (1988), through computer implementation of these algorithms (CAP, BESTFIT, BLAST N, BLAST P, FASTA and TFASTA in the Wisconsin Genetics software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, WI) or by eye examination. To the optimum To determine alignment windows, the BLAST program could be below Use of the BLOSUM 62 matrix or the PAM or PAM250 matrix with standard parameters or parameters modified to increase specificity.

"Percent of sequence identity or homology" is by comparison determined by two optimally aligned sequences over a comparison window, wherein the part of the polynucleotide sequence in the comparison window adds or deletions (i.e., vacancies) compared to the reference sequence (which does not include additions or deletions) for optimal alignment of the may include both sequences. The percentage is determined by determination the number of positions where the identical nucleic acid base or the identical amino acid residue occurs in both sequences, indicating the number of matching positions returns, dividing the number of matches Positions by the total number of positions in the comparison window and Multiply the result by 100, which is the percentage the sequence identity supplies.

The Inventors have also demonstrated that it is possible to obtain V (D) J recombination activity. by using only a fragment of the protein that is produced by the Nucleotides 39-1193 or 60-1193 is encoded. This particular fragment is also preferred in the present invention.

It It is important to note that the fragments according to the invention are preferably not completely between nucleotides 158-609, 607-660 or 29-537 of SEQ ID NO: 1 are. In fact, these nucleotides correspond to ESTs that are in the GenBank under accession numbers AA306797 (nucleotides 29-537) and AA315885 (nucleotides 158-609, 607-660). However, these EST disclosures are incomplete since AA306797 an "N" in the position 91, which makes it unclear, since it contains all 4 different nucleotides represents (the actual Nucleotide is a "C" as at the position 119 of SEQ ID NO: 1). Next AA306797 starts before the first one Methionine as identified in the present invention (nucleotide 39 of SEQ ID NO: 1), which is not the actual start codon of the protein the invention suggests. EST AA315885 possesses in comparison to the Sequence of the invention an added "C" am Nucleotide 453 of AA315885 (corresponding to nucleotide 610 of SEQ ID No. 1).

The Disclosures of AA306797 and AA315885 are by Dronkert et al. (2000, Mol. Cell. Biol., 20, 4553-61) has been used to (according to Translation of the EST) a portion of the human SNM1c protein partially homologous to the murine SNM1, the subject the revelation is. The problems with the two EST mentioned above led to a wrong translated protein.

It It is also noteworthy that Wood et al. (2001, Science, 291, 1284-9) mention, that the gene EST corresponding to SNM1C is located on chromosome 10 is, but does not specify the localization (10p), nor does it give any other information than EST AA315885, which was shown to be it is flawed.

Nucleotides 1 to 35 and 37 to 189 of SEQ ID NO: 1 have been disclosed in EST, which is disclosed in GenBank under accession number AA278590 and is incomplete and erroneous in that it has the "G" nucleotide present at position 36 of SEQ ID NO: 1 is missing.

The Nucleotides 1848 to 2321 correspond to some nucleotides found in EST present in GenBank under accession number AI859962 is revealed and incomplete is. This EST discloses a defective part of the complementary sequence SEQ ID NO: 1, the nucleotides that are complementary to the nucleotides, which are present at the start of EST AI859962, do not correspond to the last Nucleotides of SEQ ID NO: 1.

This Contains EST partly EST AA278850, which is also incomplete and faulty (mismatch a nucleotide compared to the complement of SEQ ID NO: 1).

Therefore These revelations are not only flawed and incomplete, but link not the nucleic acid either of the invention as defined above with V (D) J recombination and the SCID defect, as the inventors in the present application first names. This would have confused the professional who would not have been able to do much To obtain information from these disclosures.

The The present invention is also directed to a vector comprising the Nucleic acid molecule of the invention includes, in particular the nucleic acid sequence, the nucleotides 39-2114, 60-2114, 39-1193 or 60-1193 of SEQ ID NO: 1. Numerous vectors are known in the art, and they may be, for example Be expression vectors or amplification vectors.

The The invention is also directed to a host cell comprising the vector of the invention includes.

• a) Exprimieren des erfindungsgemäßen Nukleinsäure-Moleküls in einem geeigneten Wirt, um ein Protein zu synthetisieren, das an der V(D)J-Rekombination und/oder DNA-Reparatur beteiligt ist, und
• b) Isolieren des Proteins, das an der V(D)J-Rekombination und/oder DNA-Reparatur beteiligt ist.

The invention is also directed to a method of producing a protein involved in V (D) J recombination and / or DNA repair comprising the steps of:

• a) expressing the nucleic acid molecule of the invention in a suitable host to synthesize a protein involved in V (D) J recombination and / or DNA repair, and
• b) isolating the protein involved in V (D) J recombination and / or DNA repair.

The The present invention is also directed to an isolated nucleic acid molecule which the complement of the isolated nucleic acid molecule of the invention as above is defined, and directed to an isolated protein or peptide, that by the nucleic acid the invention is encoded. As can be seen below, the Protein or peptide of the invention either by recombinant DNA, chemical or other techniques are obtained.

The expressions Polypeptide and protein should be understood to have a specific Strand of amino acids mean, of course or can be synthetic. The person skilled in the art knows ways to vary of amino acids. Preferred proteins or polypeptides are in particular the SEQ ID No. 2 and amino acids 1-385 or 8-385 of SEQ ID NO: 2.

The Expression vectors of the invention preferably contain a promoter, Translation start and termination signals as well as suitable regions for the Regulation of transcription. They have to keep up in the host cell become. The person skilled in the art knows such vectors and the manners, proteins to produce and to clean, in particular through the use of Markers (such as the histidine marker or glutathione). It is also possible, in vitro translation kit to use that in large Scope available are to produce the protein or peptide according to the invention.

The nucleic acid of the invention or a fragment thereof can be used standard ligation techniques or homologous recombination inserted into a suitable expression or amplification vector become. The vector is chosen that it is the amplification of the nucleic acid of the invention and / or allows the expression of the gene.

The Vectors can so chosen that they are in a big one Variety of hosts, such as prokaryotic, yeast, insect (baculovirus systems) and / or eukaryotic host cells are functional. The choice of Host cell may be dependent on the characteristics of the polypeptide to be expressed or its fragment depend, For example, if post-translational modifications are required are (such as glycosylation and / or phosphorylation). If this is the case Case, eukaryotic cells such as yeast, insect or mammalian host cells are preferable.

The person skilled in the art knows various types of vectors that can be used, and the ver various techniques used to invade host cells (electroporation, lipofection ...).

An overview of the various useful vectors, host cells and regulatory DNA sequences on the vectors, depending on the host cells, can be found in the US 6,165,753 , in particular column 9, line 34 to column 13, line 36 are found. This technical part of the document, which relates to the cyclin E2 gene and polypeptide but can be generalized to any other cDNA or gene, is hereby incorporated by reference.

One further overview of the various vectors, regulatory sequences, host cells and methods for expression of polypeptides that may be used in WO 99/55730, page 6, line 3 to page 25, line 6 found which is incorporated herein by reference.

In summary For example, the vectors of the invention contain at least one selectable one Marker gene encoding a protein responsible for the survival and growth of the host cell in a selective culture medium is required. Typical selection marker genes encode proteins present in prokaryotic host cells either a resistance to antibiotics, such as ampicillin, tetracycline or Kanamycin, complement auxotrophic defects of the cell (for example, Ura yeasts). Preferred selectable markers are the kanamycin resistance gene, the ampicillin resistance gene and the Tetracycline resistance gene. The kanamycin resistance gene is preferred when vectors are present in both the prokaryotic and eukaryotic (Mammalian) cells are active, since the protein is in mammalian cells confer resistance to neomycin.

The Vectors of the invention also contain a ribosome binding sequence, such as a Shine-Dalgarno, a Kozak sequence or an internal ribosome entry site (Ires).

A Signal sequence can also be used to exclude the polypeptide of the host cell when it is synthesized. The expert For example, many signal sequences are known and each of those described in the selected Host cell is functional (homologous or heterologous), can communicate with the nucleic sequence according to the invention be used.

The Vectors of the invention are typical of an initial vector, such as a commercially available vector, derived. Such vectors can some of the elements to include in the finished vector, contain or not, such elements by suitable molecular biological Techniques introduced become. So can added Elements individually after enzymatic digestion and ligation of the element introduced into the vector become. This process is well known in the art and is for example, in Sambrook et al., supra.

preferred Vectors from which it is possible is to obtain the vectors of the invention are with bacterial, Insect and mammalian host cells compatible and close, without limitation to be pCRII, pCR3 and pcDNA3 (Invitrogen Company, San Diego, California), pBSII (Stratagene Company, LaJolla, California), pET15b (Novagen, Madison, Wisconsin), PGEX (Pharmacia Biotech, Piscataway, N.J.), pEGFP-N2 (Clontech, Palo Alto, California), PETL (BlueBacll; Invitrogen) and pFASTBacDual (Gibco / BRL, Grand Island, N.Y.).

To Construction of the Vector and Insertion of the Nucleic Acid of the Invention can be the full vector for the Amplification and / or polypeptide expression in a suitable host cell be advertised.

such Host cells can prokaryotic host cells (such as E. coli, Bacillus subtilis) or eukaryotic host cells (such as a yeast cell, an insect cell or a vertebrate cell). The host cell can if they cultured under suitable conditions, synthesize the polypeptide, which then off the culture medium (when the host cell secretes it into the medium) or directly from the host cell that produces it (if it is not is secreted) can be collected. After collecting this can Polypeptide using methods such as molecular sieve chromatography, affinity and the like are cleaned.

The choice of host cell for polypeptide production depends in part on whether the polypeptide is to be glycosylated or phosphorylated (in which case eukaryotic host cells are preferred), and on the manner in which the host cell is capable of incorporating the protein into its own native tertiary structure (eg, proper orientation of the disulfide bridges, etc.) to "fold," so that biologically active protein produced by the cell becomes. If the host cell does not synthesize the polypeptide having the biological activity of V (D) J recombination and / or DNA repair, the polypeptide may be "folded" after purification on various dialyses.

suitable mammalian cells or cell lines are well known in the art and include ovarian cells of the Chinese hamster (CHO), HeLa, HEK293, Hep-2, 3T3 cells, Monkey COS-1 and COS-7 cell lines and the CV-1 cell line, mouse neuroblastoma N2A cells, HeLa mouse L-929 cells, 3T3 lines derived from Swiss, Balb-c or NIH mice derived, BHK or NaK hamster cell lines.

helpful Bacteria host cells responsible for the present invention are suitable include the various strains E. coli, such as HB101, DN5.α., DH10 or MC1061. Different strains of B. subtilis, Pseudomonas spp. or Bacillus spp., Streptomyces spp. and the like can also be used in this process.

Lots Yeast strains those known in the art are also as host cells for expression The polypeptides of the present invention are available and the strains of Saccharomyces cerevisiae, S. pombe, Kluyveromyces ... prefer.

Of the Admission (also called "transformation" or "transfection") of the vector into the chosen one Host cell may be prepared using methods such as calcium chloride, Electroporation, microinjection, lipofection or the DEAE-dextran method be achieved. The chosen one Procedure is partly a function of the type of use to be made Host cell. These methods and other suitable methods are are well known to those skilled in the art and are e.g. in Sambrook et al., supra, specified.

Of the A person skilled in the art knows the medium to be used for the cultivation of the host cells, including the Selection agent (antibiotic) to be added to the medium, dependent from the marker located on the vector inserted into the cell.

The The amount of polypeptide produced in the host cell may be below Use of standard methods known in the art, such as, without restriction, Western blot analysis, SDS polyacrylamide gel electrophoresis, non-denaturing Gel electrophoresis, HPLC separation, immunoprecipitation and / or activity assays, such as DNA binding gel shift assays.

The Purification of the polypeptide produced by cells of the invention can if it is in solution is present (secretion or excretion), using a variety be accomplished by techniques. If there is a marker like hexahistidine contains It can essentially be done in a one-step process by passing the solution purified by an affinity column be, where the column matrix a high affinity for the Marker or directly for having the polypeptide (nickel column for polyhistidine).

If the polypeptide is prepared without attached label, others can well-known method for the cleaning can be used. Such methods include without restriction Ion exchange chromatography, molecular sieve chromatography, HPLC, native gel electrophoresis in combination with gel elution and preparative isoelectric focusing ( "Isoprime" machine / technique, Hoefer Scientific). In some cases can Two or more of these techniques are combined to give an increased purity to achieve.

If It is anticipated that the polypeptide was found mainly intracellularly can, intracellular Material (including inclusion bodies gram-negative bacteria) using any one skilled in the art known standard technology can be extracted from the host cell. For example, the host cell can by french press, homogenization and / or sonication, followed by centrifugation, lysed, to release the contents of the periplasm / cytoplasm.

If the polypeptide has formed in the periplasm inclusion body, the Polypeptide using methods known in the art obtained, in particular with the help of chaotropic agents such as guanidine or urea to release the inclusion bodies and soluble close. Further dialysis contributes to the polypeptide to solve.

It is also possible to use other standard methods well known to those skilled in the art, such as electrophoresis separation followed by electroelution, various types of chromatography (immunoaffinity, molecular sieve and / or ion exchange) and / or high pressure liquid chromatography. In some In some cases, it may be preferable to use more than one of these methods for complete purification.

In addition to Preparation and Purification of the Polypeptide Using Recombinant DNA Techniques can the polypeptides, fragments and / or derivatives thereof by chemical Synthetic method (such as solid-phase peptide synthesis) using manufactured in the art known methods, such as those those of Merrifield et al., (J. Am. Chem. Soc., 85: 2149 [1963]), Houghten et al. (Proc Natl. Acad Sci., USA, 82: 5132 [1985]) and Stewart and Young (Solid Phase Peptide Synthesis, Pierce Chemical Co., Rockford, III. [1984]). Such polypeptides can be synthesized with or without a methionine at the amino terminus. Chemically synthesized polypeptides or fragments may be mentioned under Use of methods specified in these references be oxidized to disulfide bridges too form. It is expected that these synthetic polypeptides or Fragments have a biological activity with that is comparable to the polypeptides produced recombinantly or from natural Sources are cleaned, namely an ability and thus can promote V (D) J recombination and / or DNA repair used interchangeably with recombinant or natural polypeptide become.

The Polypeptide of the invention can be derived chemically or with a Polymer are associated. The modified polypeptides according to the invention can others have pharmacological properties than the unmodified Polypeptides, such as an elevated one or reduced half-life after administration to an animal or a human, a different pharmacokinetics ...

The Polypeptides according to the invention, their fragments, variants, and / or derivatives can be used to obtain Using standard methods to make antibodies, for example after administration to an animal such as a mouse, a rat, a rabbit or a goat using a suitable adjuvant (especially complete or incomplete Freund's adjuvant).

So are antibodies, which react with the polypeptides of the invention, as well as reactive Fragments of such antibodies also included within the scope of the present invention. The antibodies can polyclonal, monoclonal, recombinant, chimeric, single-chain and / or bispecific be. In a preferred embodiment is the antibody or the fragment thereof of either human origin or is "humanized", i. made, that an immune reaction against the antibody is prevented or minimized, if it is administered to a patient.

at the antibody fragment it can be any fragment that interacts with the polypeptides of the present invention. The invention also includes the Hybridomas that are generated by the polypeptide according to the invention or a fragment thereof presented to a selected mammal as antigen, followed by fusion of cells (e.g., spleen cells) of the mammal with certain cancerous cells by known techniques, such as the technique from Köhler and Milstein (1975 Nature 256, 495), immortalized cell lines to accomplish.

The antibodies according to the invention are, for example, chimeric antibodies, humanized antibodies, Fab or F (ab ') ₂ fragments. They can be immunoconjugates or labeled antibodies.

The Inventors of the present invention have demonstrated that Artemis gene (also as SNM1C) a new V (D) J recombination and / or DNA repair factor coding for the metallo-β-lactamase superfamily belongs and whose mutations give rise to the human RS-SCID condition.

Therefore the present invention is also directed to an in vitro method for Determine the SCID type in a patient who is in it consists in analyzing in the patient the nucleic acid that is selected from the group consisting of SEQ ID No. 1, nucleotides 39-2114, 60-2114, 39-1193 and 60-1193 of SEQ ID NO: 1, wherein one mutation the nucleic acid allows classification of the SCID as a radiation-sensitive SCID.

The Mutations that are sought include point mutations that are too a non-conservative change an amino acid of the protein or to a production of a premature termination codon lead, deletions, Insertions or modifications due to changes in genomic DNA, which corresponds to SEQ ID No. 1, and the splice donor and acceptor sites, which flank the exons.

The invention is also directed to an in vitro method of diagnosis in a patient finally, a prenatal diagnosis, a condition selected from the group consisting of a SCID, a predisposition to cancer, an immunodeficiency and carrying a mutation which increases the risk that the offspring will have such a disease, which is that the nucleic acid selected from the group consisting of SEQ ID NO: 1, nucleotides 39-2114, 60-2114, 39-1193 and 60-1193 of SEQ ID NO: 1, and / or the protein derived from the Analyzed nucleic acid in the patient, wherein a mutation of the nucleic acid and / or the protein indicates an increased risk that this condition exists.

It It is also included that the diagnostic method according to the invention so performed will be that one or both of the patient's two alleles are analyzed following any mutations (point mutations, deletions, Insertions, mutations leading to incomplete splicing ...) Watching out, which lead to the production of a non-functional protein. Therefore It must be understood that the analysis of nucleic acid, the selected is selected from the group consisting of SEQ ID NO: 1, nucleotides 39-2114, 60-2114, 39-1193 and 60-1193 of SEQ ID NO: 1, directly or indirectly performed on the genomic DNA becomes.

These Diagnostic procedures are preferably performed in vitro on DNA or RNA which from cells taken from the patient.

The Diagnostic method according to the invention can performed on genomic DNA be removed from the patient for example by amplification of Exone was isolated, in particular with the primer pairs SEQ ID NO. Figure 5 to SEQ ID NO: 32 located in the introns of the Artemis gene and the amplification of the exons and exon-intron junctions enable, The inventors have shown that in some RS-SCID patients mutated. It is clear that any primer that amplifies the exons would or allow the analysis of the exon-intron junction would, in this embodiment the diagnostic method according to the invention can be used.

A Way of execution a diagnostic method according to the invention would to perform a PCR-SSCP or to sequence the amplification product to the mutations in the Artemis gene.

The ARTEMIS (or SNM1C) protein derived from the nucleic acid of the Is therefore encoded at the V (D) J recombination and / or Involved in DNA repair. Mutations in the nucleic acid, the for the expression of a non-functional Lead protein, to lead, if they occur in both patients in one allele SCID condition in the patient. It is also predicted that the properties of the ARTEMIS protein in other areas, such as of cancer therapy, can be exploited because compounds that interact with the protein in a cancer cell, contribute to it can, to sensitize the cell against an anti-tumor agent, its effect It consists of breaking down the DNA (radiation, chemical agents).

• a) In-Kontakt-Bringen der Nukleinsäure oder des Proteins mit einer Kandidaten-Verbindung und
• b) Bestimmen der Bindung zwischen der Kandidaten-Verbindung und der Nukleinsäure oder dem Protein.

The invention is therefore directed to a method of identifying a compound capable of binding to the nucleic acid or protein of the invention, comprising the steps of:

• a) contacting the nucleic acid or the protein with a candidate compound and
• b) determining the binding between the candidate compound and the nucleic acid or the protein.

The Method for assessing the binding of a compound to a nucleic acid or a protein are well known in the art and are preferred performed in vitro. One way to achieve such a goal may be to the nucleic acid or to attach the protein to a solid support to which the one to be tested Connection flowing is left, and the recovery the connection after passing on the carrier to check. By Setting parameters, it is also possible to determine the binding affinity. The connections can can also be found by other methods, including FRET, SPA ... with the compounds and nucleic acid or protein tagged are. The assay can also be performed on cells containing the nucleic acid of the Invention, for example on a vector according to the invention, and / or the protein according to the invention express. This also provides the information about the ability of the connection, through to kick the membrane and enter the cells. These cells can Bacterial cells (search for antibiotic compounds that bind to the β-lactamase fragment of the protein) or mammalian cells be.

The Invention is therefore also directed to a compound by the method described above is identified, wherein the compound to the nucleic acid or the protein of the invention binds.

Particularly preferred compounds are compounds which bind to the β-lactamase region of the protein of the invention (first 180 amino acids of SEQ ID NO: 2) or the associated b-CASP domain (amino acids 181-385 of SEQ ID NO: 2) , Such compounds may have a chemical formula corresponding to formula I or IV of WO 00/63213, wherein the formula and the corresponding part of the specification are incorporated herein by reference, WO 99/33850, WO 01/02411 or WO 00/63213 US 6,150,350 is similar, the description of the compounds being incorporated herein by reference.

at a compound identified by a method according to the invention it can be a compound with a chemical framework (chemical Compound), a lipid, a carbohydrate (sugar), a protein, a peptide, a hybrid compound, protein lipid, protein carbohydrate, Peptide-lipid, peptide carbohydrate, a protein or a peptide that has different substituents have been grafted, act.

The Foreseen chemical compounds (with a chemical framework) can or more (up to 3 or 4) cycles, in particular aromatic Cyclen, in particular containing 3 to 8 carbon atoms and all types of substituent groups (especially lower alkyl, i. having 1 to 6 carbon atoms, keto groups, alcohol groups, halogen groups ...) exhibit. The skilled person knows how different variants a compound starting from a given skeleton Grafting of these residues on the scaffold are to produce.

The Method of the invention allows also the screening, the proof and / or the identification of compounds which inhibit the biological activity of the ARTEMIS protein can. In fact, it is possible the compounds that bind to the nucleic acid or the protein and by the method according to the invention in an assay such as a complementation assay with a vector containing SEQ ID NO: 1 or the nucleotides 39-2114 of SEQ ID NO and expressing a functional protein introduced into a cell, taken from a patient suffering from RS-SCID, and the connection to their ability is tested, restoring V (D) J recombination and / or Inhibit DNA repair in the cells. The examples illustrate one such assay, which is preferably carried out in vitro.

The present invention enables so the proof, the identification and / or the screening of connections for the treatment of diseases useful could be, involving V (D) J recombination and / or DNA repair. However, the Compounds identified by the method according to the invention were, possibly be optimized to be used in a therapeutic treatment to become a superior one activity and / or lower toxicity exhibit.

• – Durchmustern von Verbindungen mit der gesuchten Aktivität bei einem relevanten Modell durch ein geeignetes Verfahren,
• – Auswahl der Verbindungen, welche die erforderlichen Eigenschaften (hier Modulation von V(D)J-Rekombination und/oder DNA-Reparatur) aufweisen, aus dem ersten Durchmusterungstest,
• – Bestimmung der Struktur (insbesondere der Sequenz (wenn möglich der tertiären Sequenz), wenn sie Peptide, Proteine oder Nukleinsäuren sind, der Formel und des Gerüsts, wenn sie chemische Verbindungen sind) der ausgewählten Verbindungen,
• – Optimieren der ausgewählten Verbindungen durch Modifikation der Struktur (zum Beispiel durch Änderung der stereochemischen Konformation (zum Beispiel Überführen der Aminosäuren in einem Peptid von L in D), Addition von Substituenten am Peptid- oder chemischen Gerüst, insbesondere durch Pfropfen von Gruppen oder Resten auf das Gerüst, Modifikation der Peptide (siehe insbesondere Gante "Peptidomimetics", in Angewandte Chemie – International Edition Engl. 1994, 33, 1699-1720),
• – Durchlaufen und Durchmustern der "optimierten" Verbindungen bei geeigneten Modellen, die häufig Modelle näher an der untersuchten Krankheit sind. In diesem Stadium würde man häufig Tiermodelle verwenden, insbesondere Nager (Ratten oder Mäuse) oder Hunde oder nicht-menschliche Primaten, die gute Modelle für SCID oder Krebse sind, und nach den phänotypischen Änderungen in den Modellen nach Verabreichung der Verbindung Ausschau halten.

In fact, the development of new drugs is often performed on the following basis:

• - screening of compounds with the sought activity on a relevant model by a suitable method,
• Selection of the compounds which have the required properties (in this case modulation of V (D) J recombination and / or DNA repair) from the first screening test,
• Determination of the structure (in particular the sequence (if possible the tertiary sequence), if they are peptides, proteins or nucleic acids, of the formula and of the skeleton, if they are chemical compounds) of the selected compounds,
• Optimizing the selected compounds by modifying the structure (for example by altering the stereochemical conformation (for example, converting the amino acids in a peptide of L into D), addition of substituents on the peptide or chemical backbone, especially by grafting groups or residues the framework, modification of the peptides (see in particular Gante "Peptidomimetics", in Angewandte Chemie - International Edition Engl. 1994, 33, 1699-1720),
• - going through and screening the "optimized" compounds on suitable models, which are often models closer to the disease being studied. At this stage, one would often use animal models, particularly rodents (rats or mice) or dogs or non-human primates, which are good models for SCID or cancers and look for the phenotypic changes in the models after administration of the compound.

The The present invention also encompasses the compounds which are to be followed the steps or equivalent Steps, as described, have been optimized.

The present invention is also applicable to any member of the nucleic acid, the vector, the host cell, the protein, the antibody and the compound of the invention as a medicament. In fact, these units may be used alone for the treatment of various types of diseases, particularly those in which V (D) J recombination and / or DNA repair is involved, the diseases being without limitation RS-SCID , Immunodeficiency, cancer. These units may also be used in combination with a further treatment suitable for the disease, the use being simultaneous, separate or sequential.

Especially can the products according to the invention in combination with cytokines, growth factors, antibiotics, anti-inflammatory Agents and / or chemotherapeutic agents are used as is for the treated indication is suitable.

The Invention is also directed to a pharmaceutical composition, a pharmaceutically acceptable excipient, carrier or a pharmaceutically acceptable diluent together with at least a member of the nucleic acid, the vector, the host cell, the protein, the antibody and the compound of the invention.

Of the A person skilled in the art knows the suitable auxiliaries and carriers that can be used, and You can, for example, water for injection, preferably with others Materials added, in solutions for administration are common to mammals, or neutral buffered saline or saline mixed with Lead serum albumin. Some carriers can in Remington's Pharmaceutical Sciences, 18th Edition, A.R. Gennaro, Hsg., Mack Publishing Company [1990].

The The pharmaceutical composition of the invention may be administered orally, nasally, via the Mucosa administered or in particular intravenously, intramuscularly or subcutaneously be injected. The carrier and / or adjuvant becomes dependent chosen by the route of administration. The already cited US patent 6,165,753 gives examples of suitable routes of administration and excipients (column 17, line 31 to column 21, line 55, the general information herein is incorporated by reference).

The Invention is also based on the use of at least one member from the nucleic acid, the vector, the host cell, the protein, the antibody, the Compound and the pharmaceutical composition of the invention for the Preparing a drug intended for the treatment of a disease where V (D) J recombination and / or DNA repair is a Plays a role, whereby the illness is selected in particular from the group, those from cancer, SCID, especially RS-SCID, immunodeficiency, for example due to problems in the switch of heavy chains of immunoglobulins or due to problems in the process of somatic hypermutation the immunoglobulins selected is.

The US 6,165,753 also describes methods of gene therapy, and this teaching is incorporated by reference.

In a preferred embodiment becomes the nucleic acid the person is administered so that it enters stem cells, i. the cells that are the producers of the cells of the immune system. This may be in vivo or ex vivo after removal of the cells of the patient, Select the stem cells, introduce the nucleic acid into the selected cells and reinjection of the transformed cells in performed to the patient become.

For the penetration the nucleic acid into the cells various means are used by the skilled person. Especially Is it possible, the nucleic acid introduce into the cells by means of a viral vector.

This Virus can be of human or non-human origin, as long as it has the ability to infect the cells of the patient. In particular, the virus selected from the group consisting of adenoviruses, retroviruses (oncoviruses such as RSV, spumaviruses, lentiviruses), poxviruses, herpesviruses (HSV, EBV, CMV ...), iridovirus, hepadnavirus (hepatitis B virus), papoviruses (SV40, Papillomavirus), parvoviruses (adeno-associated virus ...), reoviruses (Reovirus, rotavirus), togaviruses (arbovirus, alphavirus, flavivirus, Rubivirus, pestivirus), coronaviruses, paramyxoviruses, orthomyxoviruses, Rhabdoviruses (rabies virus), bunyaviruses, arenaviruses, picornaviruses (Enterovirus, Coxsackievirus, Echovirus, Rhinovirus, Aphtovirus, Cardiovirus, hepatitis A virus ...), modified Ankara virus and derived viruses thereof.

By derived viruses is meant that the virus has modifications that it adapts to humans (if it is a virus of non-human origin, the human cells without the modifications could not infect) and / or reduce its potential or actual pathogenicity. In particular, it is best if the virus used for gene transfer is replication-deficient in the human body. This is an important safety issue since control of the expression of the functional gene may be important to the implementation of the method of the invention. Nor does one desire that dissemination of the viral vector carrying the gene of therapeutic interest to other cells or to other humans takes place.

This is the reason why the viral vector used in the method of the present Invention is used, preferably replication-deficient and therefore with the aid of a helper virus or in a complementary cell line would be made, which is the genetic material necessary for producing sufficient Virustiters needed will bring in trans.

such defective viruses and suitable cell lines are in the art, for example in U.S. Patent 6,133,028, which is defective adeno-associated Viruses (AAV) and related Describes complementation cell lines and its contents here by Reference is made. Other suitable viruses will be, for example in WO 00/34497. In adenoviruses or AAV it can be interesting to delete the E1 and / or E4 region.

at the MFG virus described below, one can use the complementation Ψ-CRIP cell line, in Hacein-Bey et al. (1996, Blood 87, 3108-16), herein Reference has been made. Other suitable cell lines can also be used.

Around To improve the long-lasting effect of the correction, one would prefer a virus that integrates the functional gene into a chromosome of the infected cells.

Especially you would Adenoviruses, some of which are replication - deficient Those skilled in the art, or select retroviruses, especially those derived from mice Retroviruses. Among the retroviruses that can be used, one would a vector based on the myeloproliferative sarcoma virus (MPSV), as described in Bunting et al. (1998, Nature Medicine, 4, 58-64, the contents of which are incorporated herein by reference) described. Another well-suited retrovirus for carrying out the Method of the invention is the MFG vector, derived from the MLV virus (Moloney retrovirus) and in Hacein-Bey et al. (1996, Blood 87, 3108-16) or Cavazzana-Calvo et al. (2000, Science, 288, 669-72), the contents of which both documents are incorporated herein by reference.

The Choice of for the implementation The virus to be used in the method of the invention is a function the characteristics of the virus and the complementation cell line. It is It is clear that different viruses have different properties (especially LTR in retroviruses) and that the viruses listed above and cell lines just examples of Means are for the implementation of the method of the invention can be used, and that they are not as restrictive to be viewed. The expert knows how the best combination Gen virus cell line and / or helper virus for to choose any given situation is.

In a further embodiment becomes the nucleic acid introduced into the cell by means of a synthetic vector, which selected may be from the group consisting of a cationic amphiphile, a cationic lipid, a cationic or neutral polymer, a protic polar compound such as propylene glycol, polyethylene glycol, Glycerol, ethanol, 1-methyl-L-2-pyrrolidone or their derivatives and an aproptic polar compound such as Dimethylsulfoxide (DMSO), diethylsulfoxide, di-n-propylsulfoxide, dimethylsulfone, Sulfolane, dimethylformamide, dimethylacetamide, tetramethylurea, Acetonitrile or its derivatives. The expert in the art knows synthetic vectors that can be used and enable a high degree of transfection such as the reagents Lifofectine and Lipofectamine, available from Life Technologies (Bethesda, MD) available are.

It is predicted that the expression of the functional Artemis gene in the cells of the SCID patient a selective advantage for the cells or progeny of the cells compared to non-transformed Cells or progeny of untransformed cells brings and leads to an improvement of the condition of the patient.

By selective advantage, it is meant that the proportion of cells that have been corrected by introduction of the functional gene will increase as compared to the cells of the same type in which the gene is not functional and that this will alleviate the disease or lead yourself to a cure de.

The Possibility, to obtain such a selective advantage is for another Gen of Cavazzana-Calvo et al. (2000, Science, 288, 669-72) Service.

The Use of the invention is best performed when the cells into which the functional Artemis gene is introduced, stem cells or undifferentiated cells are, which are precursor cells of the cells, lack expression of the functional Artemis gene. This would be to the Lead the fact that a big one Number of cells is corrected over time as the offspring the stem cells would also be corrected. Next, there's the Stem cells over time differentiate into a large number of cells would just the need to have a small initial number of cells too transfect.

It is possible, some of the stem cells for the hematopoietic System to identify, since they harboring on their surface the marker CD34 (CD34 + cells). Their activation can be carried out in vivo or ex vivo (sorting of these Cells, transfection and reinfusion by intravenous injection). The expert knows that stem cells can be isolated from umbilical cord blood.

It may be interesting to induce the cell cycle of stem cells to increase the efficiency of transfection. That is in particular the Case when the method of the invention is performed on cells ex vivo.

In In fact, some of the vectors responsible for the introduction of the interesting Genes in the cells can be used, vectors that are only non-dormant Cells can be incorporated. In order for the cells to replicate, growth factors are preferably used and / or cytokines, such as CSF (GM-CSF, M-CSF, G-CSF), interleukins (preferably IL-1, 2, 3, 4, 6, 7, 8, 9, 10, 15), interferons (especially α or γ). You could too Stem cell factor, megakaryocyte differentiation factor, optionally coupled with polyethylene glycol or Flt-3-L. These factors can be alone or used in combination.

If the use of the invention is carried out ex vivo, the stem cells can also be used in a medium that is compatible with other nutrients, as serum (fetal bovine serum, as usual, or preferably fetal Cell serum) is. It may also be interesting to see the cells to be transfected in plates, the cell adhesion elements such as cell adhesion proteins (especially fibronectin or vitronectin) or contain the peptides, which have been shown to promote cell adhesion. It is clear that the Choice of culture medium by the type of cells and the desired Target is affected.

In the use according to the invention become the nucleic acid, the vector, the host cell, the protein, the antibody, the Compound and the pharmaceutical composition of the invention preferably administered together with a genotoxic agent, the use being simultaneous, separate or sequential is.

The Invention also relates to a transgenic non-human mammal, in its genome, the nucleic acid sequence the invention is integrated, in particular a nucleic acid sequence, the selected is selected from the group consisting of nucleotides 39-2114, 60-2114, 39-1193 and 60-1193 of SEQ ID NO: 1 and operatively with regulatory elements connected is, wherein the expression of the coding sequence the amount of ARTEMIS protein and / or the extent of V (D) J recombination and / or DNA repair of the mammal in the Increased compared to a non-transgenic mammal of the same species.

It it is preferred that the nucleic acid sequence present in the genome of the non-human transgenic animal, encodes a polypeptide selected from the group of polypeptides comprising amino acids 1-692, 1-385 or 8-385 of SEQ ID NO: 2. It is also preferred that the nucleic acid sequence, which integrates into the genome of the non-human transgenic animal is a polypeptide encoded by the group of polypeptides selected is that from the amino acids 1-692, 1-385 or 8-385 of SEQ ID NO: 2.

It It is also envisaged that the regulatory elements (promoters, enhancers, Introns, similar those in mammalian expression vectors can be used) can be tissue specific, what the overexpression of the ARTEMIS protein only possible in a specific type of cells. In particular knows the expert the various promoters used for this purpose can.

The Insertion of the construct into the genome of the non-human transgenic Tiers of the invention can be carried out by means of methods which are well known to those skilled in the art and can be either statistically or be targeted. In a nutshell, the expert constructs a vector containing the sequence for insertion into the genome and a Selection marker (for example, the gene encoding the protein, the Which confers resistance to neomycin), and may cause he enters the embryonic stem cells (ES) of an animal. The Cells are then selected with the selection marker and a Embryo, for example by microinjection into a blastocyst, which can be obtained by pregnant the uterus Females perfused. The reimplantation of the embryo and the choice the transformed animals, followed by the potential backcross, enable it, such non-human to get transgenic animals. To obtain a "purer" animal, the selection marker gene can be cut out using a site-specific recombinase when it is flanked by the correct sequences.

The Invention also relates to a transgenic non-human mammal, whose genome is a destruction of the endogenous Artemis gene represented by SEQ ID NO: 1 includes, wherein the destruction of the Insertion of a selectable marker sequence and wherein the destruction a non-human mammal has the result compared to a non-human wild-type mammal shows a defect in V (D) J recombination and / or DNA repair.

In a preferred embodiment is the destruction a homozygous destruction.

In a preferred embodiment has homozygous destruction a zero mutation of the endogenous gene encoding ARTEMIS.

In a preferred embodiment is the mammal a rodent, in the most preferred embodiment, is the mammal a mouse.

The The invention also encompasses an isolated nucleic acid that is an Artemis knockout construct comprising a selectable marker sequence derived from DNA sequences flanked, which are homologous to the endogenous Artemis gene whose cDNA is represented by SEQ ID NO: 1, wherein when the construct into a non-human mammal or an ancestor of the non-human mammal is introduced in the embryonic state, the selectable marker sequence is the endogenous Artemis gene in the Genome of the non-human mammal destroyed, so that the non-human mammal compared to a non-human wild-type mammal shows a defect in V (D) J recombination and / or DNA repair.

This Construct is used to obtain animals containing the destroyed copy of the Artemis gene, and is basically on a vector worn, which is also an object of the invention.

The Invention also relates to a mammalian host cell, whose genome is a destruction the endogenous Artemis gene, the destruction of the Insertion of a selectable marker sequence. Prefers is the destruction homozygous and leads to non-expression of a functional ARTEMIS protein (or Expression of a non-functional protein).

It should be noted that the destruction by known in the art Method can be obtained and may be conditional, i. only in specific types of cells is present or at some moments the Development is induced. The method to achieve such a goal can achieve the use of site-specific recombinases such as Cre (which recognizes lox sites) or FLP (which recognizes RFT sites) recombinases be under the control of a cell-specific promoter. It was shown that these recombinases (especially Cre) for modifications are suitable, and their activity can be induced by injection of a substrate (such as a hormone) become. These modifications are known in the art and may be, for example in Shibata et al. (1997, Science 278, 120-3).

Therefore may be the non-human transgenic animal or the cell of the invention possibly no longer show the selectable marker, which in the Exposure of recombinases may have been removed, which to destruction lead the gene. However, in the process of obtaining such destruction, it is selectable marker has been inserted into the Artemis gene mainly to to allow the selection of the transformed cells.

The US 6,087,555 describes a way to obtain a knock-out mouse and the general teaching of this patent is incorporated herein by reference (col. 5, line 54 to col. 10, line 30). In this Patent describes an OPG knock-out mouse, the same procedure applies to an Artemis knock-out mouse. The person skilled in the art also finds in Hogan et al. (Manipulating the Mouse Embryo: a Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1986).

In a further embodiment The invention relates to a non-human transgenic mammal whose Genome a first destruction, which is that of the endogenous Artemis gene, and includes a second destruction, which is that of the endogenous p53 gene. In the preferred embodiment is at least the first destruction or the second destruction a homozygous destruction. In the most preferred embodiment are both the first destruction as well as the second destruction both homozygous destructions, what especially a zero inactivation both the artemis and the p53 gene result.

In a preferred embodiment is the transgenic mammal a rodent, especially a rat or a mouse.

The non-human transgenic mammal can be used as a model to study pro-B-cell lymphoma become. In fact, the invention relates to a method of providing a model for the study of Pro-B-cell lymphoma, which includes that one the transgenic mammal for one Person who provides such a model for the investigation of Pro-B-cell lymphoma needed, and a method for studying pro-B-cell lymphoma, which the study of the transgenic mammal includes.

The non-human transgenic animals and the knock-out cells of the invention may also for the identification of pharmacologically interesting compounds be used. Therefore, the invention also relates to a method for screening compounds that undergo V (D) J recombination and / or DNA repair, comprising contacting a compound with the non-human mammal or the knock-out host cell of the invention and determining the increase or decrease of V (D) J recombination and / or DNA repair in the non-human mammal or the host cell compared to V (D) J recombination and / or DNA repair of non-human mammal or the host cell prior to administration of the compound.

One A method for testing the genotoxicity of compounds comprising bringing a connection with the non-human mammal or a host cell of the invention and determining the increase or decrease in V (D) J recombination and / or DNA repair in the non-human mammal or the host cell compared to V (D) J recombination and / or DNA repair of the non-human mammal or host cell before the administration of the compound is also an object the invention.

Function of Artemis

Even though from the phenotype of RS-SCID patients can be concluded that Artemis one Part of the ubiquitous Machinery at the DNA DSB (Double Strand Break) repair, in which also the V (D) J recombination process is involved is, the exact function must be for this new factor can still be determined.

The repeated search for a global homologue of Artemis in others Species in protein databases so far has no promising Candidates. The only similarity consists of the peptide 0083, which is the entire N-terminal Section of Artemis includes, to the various members the SNM1 family.

• – Erstens unterscheiden sich die drei Proteine trotz ihrer SNM1-Ähnlichkeitsregionen in den mit diesen assoziierten Domänen. Insbesondere sind die 331 Aminosäuren, die die C-terminate Region von Artemis bilden, in SNM1/PSO2 nicht vorhanden und zeigen keinerlei offensichtliche Ähnlichkeiten zu irgendeinem anderen bekannten Protein.
• – Zweitens zeigen, obwohl murine und aus Hefe stammende SNM1/PSO2-Mutanten einen starken Defekt bei der Reparatur von durch DNA-Strangvernetzungsmittel verursachten DNA-Schädigungen zeigen (Henriques und Moustacchi, 1980; Dronkert et al., 2000), diese keine erhöhte Empfindlichkeit gegen ionisierende Strahlungen, was anzeigt, dass diese beiden Proteine wahrscheinlich nicht direkt an der Reparatur von DNA-DSB (Doppelstrangbrüchen) beteiligt sind.

However, for at least two reasons, Artemis is clearly not the human ortholog of either mouse SNM1 or yeast yeast PSO2:

• First, despite their SNM1 similarity regions, the three proteins differ in their associated domains. In particular, the 331 amino acids that make up the C-terminal region of Artemis are absent in SNM1 / PSO2 and show no apparent similarity to any other known protein.
• Secondly, although murine and yeast-derived SNM1 / PSO2 mutants show a strong defect in repairing DNA damage caused by DNA strand crosslinking agents (Henriques and Moustacchi, 1980, Dronkert et al., 2000), these do not show increased sensitivity against ionizing radiation, indicating that these two proteins are unlikely to be directly involved in the repair of DNA DSB (double strand breaks).

This is in sharp contrast to the phenotype of RS-SCID patients, their primary molecular defect actually The lack of DNA DSB repair is due to the lack of formation of coding links ( "Coding joints ") in the course V (D) J recombination and the increased sensitivity of bone marrow and fibroblast cells to γ-rays (Cavazzana-Calvo et al., 1993, Nicolas et al. 1998).

Interestingly, Both Artemis, mouse SNM1 and yeast yeast PSO2 have one in common domain which is a metallo-β-lactamase folding (Aravind, 1997) and probably associated with enzymatic activity is, given the presence of almost all critical catalytic Remains (or of conserved residues, these for a function could replace). However, there is no obvious consensus on the Nature of the various metallo-β-lactamase substrates, outside from a general negatively charged composition. A sequence analysis revealed the existence of a conserved region containing the metallo-β-lactamase domain in members the Artemis / SNM1 / PSO2 subfamily (data not shown), including various other sequences associated with the nucleic acid metabolism related as two subunits of the "cleavage and polyadenylation specificity factor "(CPSF; cleavage and polyadenylation specificity factor).

It is proposed this domain, described in detail elsewhere, βCASP for metallo-β-lactamase-associated CPSF Artemis SNM1 / PSO2 domain to call. Although it is highly divergent and a majority from insertions (e.g., within yeast PSO2) this domain several conserved residues, such as the H319 in Artemis, which play a role in the reaction catalyzed by members of this subfamily could play.

It It is tempting to speculate that this domain binds to a similar substrate Way like the α-helical one domain of glyoxalase, another member of the β-lactamase family (Cameron et al., 1999).

Artemis in the NHEJ metabolic pathway of DNA repair

DNA DSB can either by homologous recombination (HR) or by the non-homologous end-joining pathway "(NHEJ; non-homologous end-linkage pathway) (summary overview in (Haber, 2000)). Although in yeast HR is the predominant Repair path is at higher Eukaryotes are mostly used and represented by NHEJ the DNA repair pathway, the while followed by a V (D) J recombination.

It It is believed that at least three protein complexes are common or one after the other at the location of the RAG1 / 2 derived DSB become. The Ku70-80 complex is likely to be the first to lesion recruited, followed by the addition of the DNA-PKcs subunit. This initial one Complex is considered the primary DNA damage sensor the DNA repair machinery activate. The XRCC4 / DNA ligase IV represents the best candidate to fill the gap indeed to repair. Recently, the RAD50 / MRE11 / NBS1 complex, the known participating in the NHEJ (Carney et al., 1998, Varon et al., 1998), found in the place of the rearranging TCR genes, what for his possible Participation in the DNA repair phase of V (D) J recombination speaks (Chen et al., 2000).

you would of course like know how Artemis plays his role in this cascade. At this Can place only speculative answers based on the analogy of phenotypes between the various deficient models, including RS-SCID, are given. It is extremely unlikely that Artemis is linked to the RAD50 / MRE11 / NBS1 complex. In fact, both lead in patients with Nijmegen as well as ataxia-telangiectasia syndrome (ATLD) mutations in the NBS1 and MRE11 gene to chromosomal instability from one induced by a profound defect in DNA damage G1 cell cycle arrest (G0 / G1 checkpoint), while V (D) spares J repair (Carney et al., 1998; Varon et al., 1998). This is in sharp Contrast to the normal G1 lock in RS-SCID fibroblasts following irradiation (unpublished Results).

• – Erstens führt ein vollständiges Null-Allel von Artemis (wie in P6, P15 und P40) nicht zu embryonaler Letalität bei Menschen. Diese Beobachtung unterstützt eine Beteilung von Artemis in dieser Phase von NHEJ nicht.
• – Zweitens ist die Neuverknüpfung von linearisierten DNA-Konstrukten, die in RS-SCID-Fibroblasten eingeführt werden, normal (unveröffentlichte Ergebnisse), während dieser Assay, wenn ein Defekt vorliegt, hochgradig diagnostisch für abnormale NHEJ in Hefe ist (Teo und Jackson, 1997; Wilson et al., 1997).

With respect to the XRCC4 / DNA ligase IV complex, there are two major differences between the mice with RS-SCID state and the XRCC4 and DNA ligase IV knock-out mice:

• - First, a complete null allele of Artemis (as in P6, P15, and P40) does not result in embryonic lethality in humans. This observation does not support an involvement of Artemis in this phase of NHEJ.
• Second, the recombination of linearized DNA constructs incorporated into RS-SCID fibroblasts normal (unpublished results), whereas this assay, if present, is highly diagnostic of abnormal NHEJ in yeast (Teo and Jackson, 1997; Wilson et al., 1997).

The Perhaps the most obvious link between Artemis and NHEJ is found with respect to the Ku / DNA-PK complex. Actually human RS-SCID patients and scid mice, which have a mutation in the DNA-PKcs coding gene, the single two known states, where one with the V (D) J recombination associated DNA repair defect alone the formation of the coding Links ("coding joints ").

The signal link ("signal joint ") - Education is in the context of a defective V (D) J recombination in all others analyzed scenarios also affected.

Furthermore The manifestations of the DNA repair defect both appear at human RS-SCID patients as well as scid mice quite strongly on the immune system limited and, for example, do not seem to have any neurological disorders or for the development of cancer, two manifestations, often with Defects in the other players of the NHEJ are associated (Roth and Gellert, 2000).

When In conclusion, the invention describes the identification and Cloning of Artemis, a New Factor of V (D) J Recombination coding gene. Mutations of Artemis cause T-B SCID defects in humans due to a lack of repair of the RAG1 / 2-mediated DNA double strand breaks. Artemis belongs to a big one Family of molecules, as part of their presumed catalytic site form a metallo-β-lactamase folding. A branch of this family, which also contains SNM1 from yeast and PSO2 from the Mouse includes, notes attached another domain, which as βCASP refers to the activity of this subgroup of proteins to nucleic acids could direct and accordingly serves the purpose of DNA repair.

Finally are probably other domains, which still need to be determined for the Conduct the various Artemis / SNM1 / PSO2 proteins to theirs specific DNA repair pathways, DSB repair for Artemis or the DNA ICL repair for SNM1 / PSO2, responsible.

The following examples are only for Illustrative purposes are intended and should not be so construed Be that the scope of the invention in any way restrict.

EXAMPLES

Example 1: Cloning the Artemis cDNA

Patients and cells

It 13 RS-SCID patients from 11 Families, due to their typical phenotype of autosomal recessive SCID at full Absence of peripheral T and B lymphocytes, but presence of natural Killer cells (Fischer et al., 1997). All patients showed impaired V (D) J recombination assay in fibroblasts and for all RS-SCID families except P16, P40, and P47 could be the Radiosensitivity status to bone marrow cells and / or Fibroblasts ((Cavazzana-Calvo et al., 1993, Nicolas et al., 1998; Moshous et al., 2000) and our unpublished ones Results). Genotyping of blood relatives under Use of polymorphic microsatellite markers, as elsewhere (Moshous et al., 2000) agreed with us in every case previously described localization of RS-SCID on the chromosome 10p match. The study was based on four French patients Origin (P1, P3, P6 and P15) and one of them (P15) was from one of his parents, one of Italian origin (P16), a Greek (P4) and an African (P2). The remaining patients are from four blood related Turkish Families, two of which are related (P38, P5, P11, and P12). Prior to this investigation, the families agreed for information. Skin biopsies revealed primary fibroblast cell lines derived and pseudo-immortalized with SV40, as described elsewhere (Nicolas et al., 1998), and in RPMI 1640 (GIBCO BRL), supplemented with 15% fetal Calf serum, cultured.

cloning of Artemis cDNA and amplification of genomic DNA

Starting from fibroblast RNA, first strand cDNA was synthesized. Artemis in full country The coding sequence was amplified by polymerase chain reaction (PCR) on cDNA using the Advantage-GC cDNA PCR Kit (Clontech) according to the manufacturer's recommendations and primers 0083F1 (5'-GATCGGCGGCGCTATGAGTT-3 ', SEQ ID NO: 3) and 169F (5'-TGTCATCTCTGTGCAGGTTT-3 ', SEQ ID NO: 4) designed from the EST sequences AA278590 and AI859962. The PCR products were sequenced directly on an ABI377 sequencer (Perkin-Elmer) using the BigDye Terminator Cycle Sequencing Ready Reaction (Applied Biosystems) with a series of internal oligonucleotides. Because of several alternatively spliced transcripts, sequencing was also performed on cloned PCR products. Artemis full-length cDNA was subcloned into pIRES-EGFP (Clontech) for subsequent use in transfection experiments. The genomic structure of the Artemis gene was resolved by alignment (homology-based alignment) of the cDNA sequence against the designed sequence of the Bac 2K17 (AL360083). For the specific amplification of each exon, a series of oligonucleotide primer pairs were designed. Exon 4- and exon 5 PCR products were cloned into pGemT (Promega) for subsequent use in a Southern blot analysis (see below).

The Primer for a genomic amplification of the various exons 1 to 14 SEQ ID NO: 5 to SEQ ID NO: 32. These Enable pairs of primers the amplification of each exon as indicated in the Sequence Listing, where the number of the exon is after the "Ex", "F" means forward and "R" backwards.

Results

As part of its efforts to sequencing human chromosome 10, the "Chromosome 10 Mapping Group" at the Sanger Center has constructed several artificial bacterial chromosomal (BACs) antigens spanning the entire chromosome 10 (http://www.sanger.ac .uk / HGP / Chr10), two of these contigs, 10ctg1105 and 10ctg23 ( 1A ) were of particular interest as they included several BACs that displayed the RS-SCID region flanking markers D10S1664 and D10S674 as well as D10S191 and D10S1653, which showed maximal pairwise LOD scores in A-SCID populations (Li et al ., 1998).

It was a systematic review of Nucleotide sequences corresponding to those described in the two of the "Human Chromosome 10 Sequencing Group "on Sanger Center published Contigs (http://webace.sanger.ac.uk/cgibin/ace/simple/10ace) existing 24 BACs were initiated. The analyzes were based on a silico nucleotide sequence assessment with GENESCAN (Burge and Karlin, 1997) (http://bioweb.pasteur.fr./seganal/interfaces/genscan.html) and FGENESH (Salamov and Solovyev, 2000) (http://genomic.sanger.ac.uk/gf/gfb.html) software; two programs aimed at identifying putative peptide-coding Genes in big ones to search for genomic DNA sequences. All predicted peptides were subsequently opposite the translated non-redundant GENBANK / EMBL nucleotide databases Use of TBLASTN checked. at the publication the AL360083 design sequence, which came from the BAC 2K17, said FGENESH a 149 amino acids (aa; AS) long peptide (hereinafter referred to as peptide "0083") within which 121 aa is equal to 35% and 31% respectively the proteins MuSNM1 (AAF64472) and PSO2 from yeast (P30620). The same peptide prediction was made using GENESCAN receive.

SNM1 and PSO2 are two proteins involved in the repair of DNA damage by interstrand cross-linking agents (ICL), including cisplatin, Mitomycin C or cyclophosphamide ((Henriques and Moustacchi, 1980; Dronkert et al., 2000) and references cited therein) be involved. Mouse and yeast mutants for SNM1 / PSO2 are opposite several agents that cause ICLs, hypersensitive, but not towards γ-rays, in contrast to RS-SCID patients with some bone marrow cells and fibroblasts a hypersensitivity to γ-rays was detected. The suspected Peptide encoding "0083" gene due to its chromosomal location and the similarity of the "0083" peptide to DNA repair proteins despite the significant discrepancy in the radiosensitivity phenotype good candidates. The validity of the putative "0083" peptide was determined by searching for RNA transcripts encoding this peptide in the "TIGR humane Gene Indices' database using TBLASTN (http://www.tigr.org/docs/tigrscripts/nhgi_scripts/tgi_blast.pl?organism=Human) was searched.

This query yielded the THC535641 index, within which AA278590 represented the most 5 'sequence corresponding to the I.MA.GE clone 703546. The nucleotide sequence of the opposite end of this clone (AA278850) was consistent with the THC483503 index. The AI859962 nucleotide sequence in this second index yielded the 3'th most extent of the cDNA encoding the "0083" peptide A full length cDNA was prepared by RT-PCR amplification of fibroblast RNA using the nucleotide sequences AA278590 and AA278590, respectively. This cDNA was directly sequenced and cloned into pGemT Several clones were non-productive transcripts generated by alternative splicing events (data not shown) The inventors focused on a set of clones The full length cDNA sequence of 2354 bp (SEQ ID NO: 1) contains an open reading frame (ORF) of 2079 bp. It is assumed that the ATG at position 39 or the ATG at position 60 are the Translational start site represents, based on the analysis of surrounding nucleotides, which with the Kozack consensus sequence for the initiation of Tr anslation match. The encoded protein of 692 or 685 aa designated Artemis (which corresponds to SNM1C) has a predicted molecular weight of approximately 78 kD. The "0083" peptide (which corresponds to the peptide encoded by nucleotides 348 to 800 of SEQ ID NO: 1) corresponds to I106-H254 (beginning at n. 39) and is part of a larger region which shows similarity to scPSO2 and muSNM1 ,

It can not, Completely ensure that this cDNA represents the full-length sequence, although several attempts continue the 5 'sequence to expand, failed. The polyA tail in the 3'-untranslated Region of the sequence SEQ ID NO: 1 is represented by the genomic sequence coded and is not the result of RNA processing, which is close suggests that this cDNA may be further downstream extends. Functional complementation studies (see below) strongly suggest, however, that the complete Artemis ORF is actually cloned has been. The structure of the Artemis gene (Table 1) was determined by comparison the cDNA sequence with the genomic (AL360083) sequence derived. Artemis consists of 14 exons with sizes ranging from 52 bp to 1160 bp rich. In various Artemis cDNA clones, four alternative Identified and lead exons to non-productive splices (Data not shown).

Table 1: Exon borders of the ARTEMIS gene

RS-SCID is previously a 6.5 cM sized chromosomal region formed by the polymorphic marker D10S1664 and D10S674, a region responsible for classical cDNA selection studies (Li et al., 1998; Moshous et al., 2000) is too large. A in silico evaluation of designed genomic sequences, which span this region, led the inventors to the identification of the Artemis gene.

Example 2: Expression from Artemis

Southern blot analysis

Ten μg of high molecular weight DNA was digested with HindIII or Eco88I, on a Separated 0.7% agarose gel, blotted under vacuum on a nylon membrane (Genescreen) and hybridized with P32-labeled Artemis exon 5 and exon 4-specific probes.

RNA expression analysis

PCR-ready multi-tissue cDNA was purchased from Clontech and amplified using the Artemis-specific primers 0083F4 (5'-AGCCAAAGTATAAACCACTG-3 ', SEQ ID NO: 33) and 169F (SEQ ID NO: 4) or the GAPDH supplied by the manufacturer. Primers were amplified as a control and separated on 1% agarose gels. Artemis-specific PCR products were blotted onto a nylon membrane and hybridized with an internal P ³² -labeled oligo-nucleotide, whereas the GAPDH-PCR control was visualized by ethidium bromide staining.

Results

Increased radiation sensitivity of RS-SCID versus γ-rays is not limited to the cells of the immune system, but is also a characteristic of fibroblasts, suggesting that Artemis ubiquitously expresses becomes. This was done by a PCR analysis on a group of 15 cDNAs, which are a wide range of hematopoietic and nonhemopoietic Represented tissues, approved.

The Extent of Artemis expression is ubiquitous, but weak and needed 30 PCR cycles (38 cycles for skeletal muscle) to give a suitable P32-labeled oligonucleotide Signal compared to the strong ethidium bromide staining, the for the Control gene GAPDH was obtained.

The only to a small extent Expression of Artemis could reflect a general property of the SNM1 protein family, since it was found that the basic expression of mSNM1 in ES cells was also very low (Dronkert et al, 2000). It is Note that compared to other tissues, Artemis expression in the thymus or bone marrow, the sites of V (D) J recombination elevated was.

As given the generalized increased radiation sensitivity in RS-SCID patient cells (Cavazzana-Calvo et al., 1993), Artemis showed a pleiotropic expression pattern.

Example 3: The Artemis gene is mutated in human RS-SCIDs

mutation analysis

Artemis mutation sequence analysis were either to cDNA after RT-PCR amplification or to genomic DNA after exon-specific PCR amplification executed. All PCR products were made using the BigDyeTerminator Cycle Sequencing Ready Reaction sequenced directly.

Results

The structure and sequence of the Artemis gene were analyzed in a series of 11 RS-SCID families, which included 13 patients (Table 2 and Figs 1 ). Three patients (P6, P15 and P40) were characterized by a complete lack of the Artemis transcript caused by a genomic deletion extending from exon 1 to 4. This mutation can be considered as a complete null allele.

The same genomic deletion was present on one allele in P1, the one C279T nucleotide change wore on the other allele, leading to the formation of a non-sense codon led at R74. A homozygous C279T mutation was also present in P2 and became found heterozygous at P4.

For this Series of RS-SCID patients were two other genomic deletions characteristic. A homozygous deletion spanning exons 5 to 8 at P47 led to the formation of a cDNA in which exon 4 in the grid on exon 9 spliced was what a suspected Protein lacking K96 to Q219 results. At P3 led one heterozygous genomic deletion of exons 5 and 6 to the outside the raster splicing from exon 4 to 7, resulting in a frame shift in K96. The the second allele carried a heterozygous G in this patient C nucleotide change in the canonical splice donor sequence of Exon 11, which is the outside the raster splicing from exon 10 to 12, which results in a grid shift at T300 leads, caused.

When most recently, three other splice donor sequence mutations were identified in six patients. A heterozygous G versus A nucleotide change in the splice donor site of exon 10 in P4 caused the production of a cDNA where the fusion from exon 9 to 12 the open reading frame was conserved and potentially added production of a protein lacking A261 to E317. A Homozygous G vs. T mutation in the exon 5 donor site was added the siblings P5, P11 and P12 as well as P38 found which the outside the raster splicing from exon 4 to 6 and the generation of a frame shift at K96 caused. Although this form of cDNA using exon 5 as a Result of an alternative splice event is missing, even in in RNA was detected from normal cells (data not shown), she was for all cDNAs in P5 and P38 responsible. In the patient caused P16 a homozygous deletion of G818 in exon 9 together with a homozygous C- against T-change nine nucleotides downstream in the intron, the formation of a frame shift at A254.

When Ever samples were available from the parents of the patients, they were on tested for the presence of the mutations. This could be the inheritance the mutations in P5, P11 and P12 as well as P38 and P16 by direct Sequencing of exon-specific genomic PCR products, obtained from the parents' DNA, (data not shown) confirm that coincides with autosomal recessive inheritance.

• *: Stop-Codon

In summary, all of the 13 RS-SCID patients tested in this series carry homozygous or heterozygous mutations in the Artemis gene. None of these mutations were simple missense mutations and one of them (genomic deletion of exon 1 to 4) can be considered a true null allele given the complete absence of Artemis transcript in P6, P15 and P40. All mutations will be in 1 and Table 2 recapitulates. Table 2: Mutations of the Artemis gene in RS-SCID patients

• *: Stop codon

Of the first indication that Artemis actually participated in RS-SCID Gen was, came from the identification of mutations in several Patients. In total, 8 different mutations of the gene found at 11 families. Although some of the mutations recur were, it was not possible any unique correlation with the geographical origin to determine the patient.

• – Erstens umfassen drei der identifizierten Modifikationen genomische Deletionen, welche mehrere Exons überspannen, welche zu Rasterverschiebung und Auftreten einer vorzeitigen Termination in zwei Fällen und einer im Raster erfolgenden Deletion von 216 aa in einem Falle führen. Dies zeigt an, dass das Artemis-Gen möglicherweise einen Hot-Spot für eine Gendeletion darstellen könnte.
• – Zweitens besteht keine der Mutationen in einfachen Nucleotidsubstitutionen, welche Aminosäure-Veränderungen erzeugen, und nur eine, die C279T-Transversion, erzeugt eine Nonsense-Mutation (Nicht-Sinn-Mutation). Die anderen Nucleotidveränderungen betreffen Spleißdonorsequenzen, was in drei Fällen entweder zu Rasterverschiebungen oder in einem Falle zu einer im Raster erfolgenden Deletion eines Teils des Proteins führt.
• – Drittens umfasst bei drei Patienten (P5, P15 und P40) die genomische Deletion Exon 1 bis 4 und führt zu einem vollständigen Fehlen von Artemis codierender cDNA. Diese Deletion, die so aufgefasst werden kann, dass sie zu einem Null-Allel führt, demonstriert dementsprechend, dass Artemis kein essentielles Protein für die Lebensfähigkeit ist im Gegensatz zu beispielsweise XRCC4 und DNA-Ligase IV (Barnes et al., 1998; Frank et al., 1998; Gao et al., 1998) oder dass es teilweise redundant ist.

The analysis of these mutations reveals several interesting features:

• First, three of the identified modifications involve genomic deletions spanning multiple exons leading to frame shift and premature termination in two cases and an in-frame deletion of 216 aa in one case. This indicates that the Artemis gene might possibly be a hot spot for a gene deletion.
• Second, none of the mutations exist in simple nucleotide substitutions that produce amino acid changes, and only one, the C279T transversion, produces a nonsense (non-sense mutation) mutation. The other nucleotide changes involve splice donor sequences, which in either case results in either frameshifting or, in one case, in-frame deletion of a portion of the protein.
• Third, in three patients (P5, P15 and P40) the genomic deletion comprises exon 1 to 4 and results in a complete lack of Artemis encoding cDNA. Accordingly, this deletion, which can be considered to result in a null allele, demonstrates that Artemis is not an essential protein for viability as opposed to, for example, XRCC4 and DNA ligase IV (Barnes et al., 1998, Frank et al., 1998; Gao et al., 1998) or that it is partially redundant.

These Information is of particular interest in the context of a murine Knockout counterpart to the human RS-SCID condition. The participation of Artemis in the RS-SCID condition was unambiguously determined by complementation of the V (D) J recombination defect in fibroblasts of patients after transfection with an Artemis wt cDNA (next Example).

Example 4: Artemis complements the RS-SCID-V (D) J recombination defect

V (D) J recombination assay-

The V (D) J recombination assay was performed as previously described (Nicolas et al., 1998). Briefly, 5 × 10 ⁶ exponentially proliferating SV40 transformed skin fibroblasts in 400 μl culture medium (RPMI 1640, 10% FCS) were electroporated with 6 μg RAG-1 and 4.8 μg RAG-2-encoding expression plasmid together with 2 , 5 μg of the extrachromosomal V (D) J substrates pHRecCJ (coding joint) or pHRecCJ (signal joint). These plasmids carry a LacZ gene which is disrupted by a DNA stuffer sequence flanked by V (D) J recombination signal sequences. After recombination, the stuffer DNA is excised and the LacZ gene reassembled creating blue bacterial colonies when plated on XgaI / IPTG medium. pARTE-ires-EGFP (2.5 μg) was added for complementation analysis. Constructs used for transfection were recovered after 48 hours, re-introduced into DH10B bacteria and plated on plates containing XgaI / IPTG. The recombination percentage was determined by counting blue and white colonies and calculating the ratio. Blue colonies were randomly picked and the plasmid DNA sequenced to analyze the quality of the V (D) J junctions.

Results

The Inventors previously have the absence of a V (D) J recombination Derivative formation of coding joints in fibroblasts of RS-SCID patients after transfection with RAG1 and RAG2 expression constructs together with extrachromosomal V (D) J recombination substrates used for analysis the coding (pHRecCJ) linkage are specific (Nicolas et al., 1998). In contrast, was For RS-SCID fibroblasts, the signal-joint formation always as normal ((Nicolas et al., 1998) and Table 3).

The Artemis gene was added to the mammalian expression vector Plres-EGFP cloned and its functional complementation activity in the V (D) J recombination assay- in fibroblasts from 7 RS-SCID patients using the pHRecCJ substrate determined (Table 3). In all cases were after transfections in the presence of wt-Artemis blue bacterial colonies what the RAG1 / 2-driven recombination of the substrate certified while in the absence of exogenous wt artemis, virtually no such Colonies were obtained.

The frequencies of recombination events ranged from 1.5 x 10 ^-3 to 2.9 x 10 ^-3 , which was consistent with the frequency of 3.2 x 10 ^{-3 obtained} using a control fibroblast cell line. Sequence analysis of the recovered pHRecCJ plasmids that formed blue colonies in this assay in fibroblast lines of P1 and P40 showed that the junctions produce bona fide V (D) J coding joints with limited tailoring of the coding ends similar to those obtained in control fibroblasts (not shown).

Taken together, these results indicate that the V (D) J recombination defect in RS-SCID in directly related to the described mutations in the Artemis gene stands and through the introduction a wt Artemis cDNA into the fibroblasts of the patients can be. Although a temporary one Expression of Artemis at a high level did not appear to be toxic No stable transfectants could be derived to the Complementation of hypersensitivity to ionizing radiation analyze. this could on a toxicity an expression of wt-Artemis at a high level over be due for a long time in the transfected fibroblasts. An analogous cellular toxicity was previously after overexpression from other human or mouse homologs of SNM1 has been described in vitro and may be a characteristic of this Be family of proteins (Dronkert et al., 2000). This is synonymous in accordance with the low level physiological RNA expression of these Genes (see above).

It It is interesting to note that this is from the first 385 amino acids of SEQ ID NO: 2 was also capable of the V (D) J recombination defect to complement in this assay.

Example 5: Artemis belongs to the Metallo-β-lactamase superfamily

Data base searches using the BLAST2 program with the Artemis AS sequence as a query revealed about the first 360 amino acids from Artemis significant similarities to several proteins, including the proteins PSO2 off Yeast and SNM1 from the mouse. Subsequent iterations with the PSI-BLAST program raised significant similarities the first 150 amino acids to well-established members of the metallo-β-lactamase superfamily.

The Metallo-β-lactamase fold, the first time for the β-lactamase from Bacillus cereus (Carfi et al., 1995), is widely used by various metalloenzymes Distribution and broad substrate specificity (Aravind, 1997). It consists of a four-layer β-sandwich with two mixed β-sheets, the by α-helices be flanked, with the metal binding sites on one edge of the β-sandwich are localized. Sequence analysis as well as a secondary structure prediction for Artemis clearly showed the conservation of for metallo-β-lactamase folding typical motifs. This is especially true for amino acids D17, [HXHKDH] 33-38, H115, and D316 participating in the metal binding pocket and represent the catalytic site of the metallo-β-lactamases, to. The last metal-binding residue of metallo-β-lactamases, H225 in Tenotrophomonas maltophilia-metallo-β-lactamase (1SML) located at the end of a β-sheet (leaflet β12), missing from Artemis / SNM1 / PSO2, could but by the aspartic acid D165 from Artemis, which is also conserved at SNM1 / PSO2, functional be replaced. The latter remainder is at the end of a predicted β-sheet, separated by an α-helix from the strand carrying the preceding metal-binding moiety.

Taken together, Not only does this analysis indicate that Artemis is likely to undergo β-lactamase folding has accepted, but possibly could also have conserved an associated catalytic activity in the Unlike many other proteins, many of the catalytic Leftovers have lost (Aravind, 1997).

Example 6: In Vitro Mutagenesis of the Artemis gene

The Analysis of the protein sequence revealed by Artemis revealed the existence of a suspected Metallo-β-lactamase domain (M1 bis R179), which suggests that Artemis has some catalytic function, such as Hydrolase, could have. This domain follows another domain (E180 to S385), referred to as β-CASP for β-lactamase CPSF-Artemis SNM1-PSO2 associated domain "(associated with β-lactamase CPSF, Artemis, SNM1, PSO2 Domain) was designated. The β-CASP domain is in a series of proteins with a function in the context of the substance change of nucleic acids (DNA repair, RNA processing ...) always associated with the β-lactone domain. After all includes the last domain (C-ter) E386 to T692. The role and interdependence of these two domains were analyzed in vitro by generating mutants and their activity tested for both V (D) J recombination and DNA repair has been.

Results

V (D) J recombination

Of the Assay is based on transfection of a fibroblast with Rag1 and Rag2 expression constructs, to the V (D) J recombination of an extrachromosomal substrate (pHRec-CJ or pHRec-CS, see Example 4). The βLact + βCASP region (M1 to S385) is sufficient to detect the V (D) J recombination defect in fibroblasts from Artemis-deficient (RS-SCID) patients.

however complements the βLact domain (M1 bis R179) alone this defect not. There is also no activity when βCASP-C (E180 to T692) or C-ter (E386 to T692) configurations of Artemis be used.

The catalytic site of metallo-β-lactamases in bacteria is conserved by the arrangement of several His and Asp residues that bind Zn atoms and confer hydrolytic activity, characterized. Many of these residues are also conserved in Artemis, what else the possible Hydrolase activity of Artemis suggests.

Several of these His and Asp residues were mutated to Val and the remainder Function of the protein was analyzed in the V (D) J assay.

D17A, H35A, D37A and D136A nearly destroyed the function of Artemis Completely, while H165A and H319A its activity reduced. H38A and H151A seem to have no effect.

DNA repair

This assay is based on analyzing the sensitivity of a retroviral vector expressing various forms of Artemis to transduced RS-SCID fibroblasts over γ-rays. While βLact-βCASP / C-ter complements the excessive radiation sensitivity of RS-SCID cells, βLact-βCASP has no effect.

Conclusions

These Mutagenesis experiments suggest that Artemis is likely to have some catalytic activity as due to its homology to bacterial metallo-β-lactamases speculated becomes.

The βLact + βCASP domain of Artemis carries his catalytic activity, da 1) they undergo V (D) J recombination on extrachromosomal substrates and 2) a mutation of several suspected ones catalytic residues (His and Asp) can destroy the function.

Nevertheless it is interesting to note that the βLact + βCASP domain is not sufficient per se seems to be Artemis activity in the context of the entire chromosome to enable because the full-length artemis protein or at least part of the C-terminal domain appears to be required, to complement the radiation sensitivity phenotype.

This suggests that Artemis is likely to interact with other proteins in interacts with the context of its substrate within the chromatin.

Based on this result it is possible that the V (D) J recombination activity towards endogenous (located in chromatin opposite extrachromosomal) IgG and TCR genes also all the Artemis protein could require.

These Situation is similar in some ways to that of the Rag2 protein. Rag2 requires a core region and sufficient to facilitate the recombination of extrachromosomal substrates but is ineffective in the rearrangement of endogenous loci.

Example 7: Analysis of Patients with partial artemis deficiency

observation

at In an assessment of SCID patients, the inventors met 4 Cases (at 2 families) that had a complex phenotype, which is ataxia telangiectasia, yet without clear ataxia. These patients suffered under severe lymphopenia and hypogammaglobulinemia.

at Some of these patients submitted the finding of chromosome aberrations in the case of lymphocytes, suggest that they have a DNA repair defect could, what continue to operate was by hypersensitivity of bone marrow to γ-rays has been.

In Fibroblasts from two of these patients (which are the two families represented) V (D) J recombination was either absent or severe decreased and was brought to the normal level by adding restores wt-Artemis, as in Example 4.

When last was it possible to show that the Artemis gene was mutated in both cases, leading to premature Stop codons at T432 and D451 (at the beginning of the C-Ter domain) resulted.

These Observation confirmed somehow the results of in vitro mutagenesis (see example 6), indicating that an Artemis protein lacking the complete C-Ter domain still have recombination activity in the chromatin context in vivo can (these patients actually have some lymphocytes), but the efficiency against endogenous loci is very weak is (these patients are severely lymphopenic).

at Two patients from the first family were immune deficient the development of a very aggressive and disseminated lymphoproliferative Disease (SLP) associated with the presence of EBV.

These SLPs can probably based on their clonality and their aggressiveness with true B-cell lymphomas be compared.

conclusion

The primary Conclusion from this observation is that Artemis probably be regarded as a "caretaker" can, as a lack of this obviously with the development of B-cell lymphoma is linked.

These Idea is supported by the literature that (in several reports) shows that defects on other factors of V (D) J recombination / DNA repair (such as Ku80, DNA-PK, XRCC4, DNA ligase IV) in animal models are always under development lead to pro-B-cell lymphoma, if they are introduced in front of a P53 / background, what they are considered true Guardian revealed.

These Hypothesis can be experimentally tested in an animal model.

BIBLIOGRAPHY

• Aravind, L. (1997). An evolutionary classification of the matallo-β-lactamase fold. In Silico Biology.
• Barnes et al. (1998). Targeted disruption of the gene encoding DNA ligase IV leads to lethality in embryonic mice. Curr Biol 31, 1395-1398.
• Biedermann et al. (1991). Scid mutation in mice confers hypersensitivity to ionizing radiation and a deficiency in DNA double-strand break repair. Proc. Natl. Acad. Sci. 88, 1394-1397.
• Blunt et al. (1996). Identification of a nonsense mutation in the carboxyl-terminal region of DNA-dependent protein kinase catalytic subunit in the scid mouse. Proc. Natl. Acad. Sci. USA 93, 10285-10290.
• Bosma et al. (1983). A severe combined immunodeficiency mutation in the mouse. Nature 301, 527-30.
• Bunge, C. and Karlin, S. (1997). Prediction of complete genes structures in human genomic DNA. J Mol Biol 268, 78-94.
• Cameron et al. (1999). Crystal structure of human glyoxalase II and its complex with a glutathione thiolester substrate analogue. Structure Fold Des 7,1067-78.
• Carfi et al. (1995). The 3-D structure of a zinc metallo-beta-lactamase from Bacillus cereus reveals a new type of protein fold. Embo J 14, 4914-21.
• Carney et al. (1998). The hMre11 / hRad50 protein complex and Nijmegen breakage syndrome: leftage of double-strand break repair to the cellular DNA damage response. Cell 93, 477-86.
• Cavazzana-Calvo et al. (1993). Increased radiosensitivity of granulocyte macrophage colony-forming units and skin fibroblasts in human autosomal recessive Severe Combined Immunodeficiency. J. Clin. Invest. 91, 1214-1218.
• Chen et al. (2000). Response to RAG-mediated V (D) J cleavage by NBS1 and gamma-H2AX. Science 290, 1962-5.
• Corneo et al. (2000). 3D clustering of human RAG2 gene mutations in Severe Combined Immune Deficiency (SCID). J. Biol. Chem. 275, 12672-12675.
• Danska et al. (1996). Biochemical and genetic defects in the DNA-dependent protein kinase in murine scid lymphocytes. Mol Cell Biol. 16, 5507-17.
• Dronkert et al. (2000). Disruption of mouse SNM1 causes increased sensitivity to the DNA interstrand cross-linking agent mitomycin C. Mol Cell Biol 20, 4553-61.
• Eastman et al. (1996). Initiation of V (D) J recombination in vitro obeying the 12/23 rule, nature 380, 85-88.
• Fischer et al. (1997). Naturally occurring primary deficiencies of the immune system. Annu. Rev. Immunol. 75, 93-124.
• Frank et al. (1998). Late embryonic lethality and impaired V (D) J recombination in mice varnishing DNA ligase IV. Nature 396, 173-7.
• Fugmann et al. (2000). Identification of two catalytic residues in RAG1 that defines a single active site within the RAG1 / RAG2 protein complex. Mol Cell 5, 97-107.
• Fulop, G.M. and Phillips, R.A. (1990). The scid mutation in Mice causes a general defect in DNA repair. Nature 347, 479-482.
• Gao et al. (1998). A targeted DNA-PKcs null mutation reveals DNA-PK independent functions for KU in V (D) J recombination. Immunity 9, 367-76.
• Gao et al. (1998). A critical role for DNA end-joining proteins in both lymphogenesis and neurogenesis. Cell 95, 891-902.
• Gottlieb, T.M. and Jackson, S.P. (1993). The DNA-dependent protein kinase: requirement for DNA ends and association with Ku antigen. Cell 72, 132-142.
• Haber, J.E. (2000). Partners and pathways repairing a double beach break. Trends Genet 16, 259-64.
• Hendrickson et al. (1991). A left between double-beach break related repair and V (D) J recombination: the scid mutation. Proc. Natl. Acad. Sci. USA 88, 4061-4065.
• Henriques, J.A. and Moustacchi, E. (1980). Isolation and characterization of pso mutants sensitive to photo-addition of psoralen derivatives in Saccharomyces cerevisiae. Genetics 95, 273-88.
• Hu, D.C., Gahagan, S., Wara, D.W., Hayward, A. and Cowan, M.J. (1988). Congenital severe combined immunodeficiency disease (SCID) in American Indians. Pediatr. Res. 24, 239.
• Jackson, S.P. and Jeggo, P.A. (1995). DNA double-strand break repair and V (D) J recombination: involvement of DNA-PK. Trends Biochem Sci 20, 412-5.
• Jhappan, C, Morse, H.C.R., Fleischmann, R.D., Gottesman, M.M. and Merlino, G. (1997). DNA-PKcs: a T-cell tumor suppressor encoded at the mouse scid locus. Nat Genet 17, 483-6.
• Junop, M.S., Modesti, M., Guarne, A., Ghirlando, R., Gellert, M. and Yang, W. (2000). Crystal structure of the xrcc4 DNA repair protein and implications for end-joining. Embo J 19, 5962-70.
• Kim, D.R., Dai, Y., Mundy, C.L., Yang, W. and Oettinger, M.A. (1999). Mutations of acidic residues in RAG1 define the active site of the V (D) J recombinase. Genes Dev 13, 3070-80.
• Landree, M.A., Wibbenmeyer, J.A. and Roth, D.B. (1999). mutational analysis of RAG1 and RAG2 identifies three catalytic amino acids in RAG1 critical for both cleavage steps of V (D) J recombination. Genes Dev 13, 3059-69.
• Li, et al. (1998). The gene for severe combined immunodeficiency Athabascan-speaking Native Americans is located on chromosomes 10p. At J Hum Genet 62, 136-44.
• Li et al. (1995). The Xrcc4 Gene Encodes a Novel Protein Involved In DNA Double-Strand Break Repair and V (D) J Recombination. Cell 83, 1079-1089.
• McBlane et al. (1995). Cleavage at a V (D) J recombination signal requires only RAG1 and RAG2 proteins and occurs in two steps. Cell 83, 387-95.
• Mombaerts et al. (1992). RAG-1 deficient mice have no mature B and T lymphocytes. Cell 68, 869-877.
• Moshous et al. (2000). A new gene involved in DNA double-strand break repair and V (D) J recombination is located on human chromosomes 10p. Hum Mol Genet 9, 583-588.
• Nicolas et al. (1996). Lack of detectable defect in DNA double-strand break repair and DNA-dependent protein kinase activity in radiosensitive human severe combined immunodeficiency fibroblasts. Eur. J. Immunol. 26, 1118-1122.
• Nicolas et al. (1998). A human SCID condition with increased sensitivity to ionizing radiations and impaired V (D) J rearrangements defines a new DNA Recombination / Repair deficiency. J. Exp. Med 188, 627-634.
• Nussenzweig et al. (1996). Requirement for Ku80 in growth and Immunoglobulin V (D) J recombination, Nature 382, 551-555.
• Oettinger et al. (1990). RAG-1 and RAG-2, adjacent genes that synergistically activate V (D) J recombination. Science 248, 1517-1523.
• Paull et al. (2000). A critical role for histone H2AX in recruitment of repair factors to nuclear foci after DNA damage. Curr Biol 10, 886-95.
• Robins, P. and Lindahl, T. (1996). DNA ligase IV from HeLa cell nuclei. J Biol Chem 271, 24257-61.
• Rogakou et al. (1999). Megabase chromatin domains involved in DNA double-strand breaks in vivo. J Cell Biol 146, 905-16.
• Rogakou et al. (1998). DNA double-stranded breaks induce histone H2AX phosphorylation on serine 139. J Biol Chem 273, 5858-68.
• Roth, D.B. and Craig, N.L. (1998). VDJ recombination: a transposase goes to work. Cell 94, 411-4.
• Roth, D.B. and Gellert, M. (2000). New guardians of the genome. Nature 404, 823-5.
• Roth et al. (1992). V (D) J recombination: Broken DNA molecules with covalently sealed (hairpin) coding ends in scid mouse thymocytes. Cell 70, 983-991.
• Salamov, A.A. and Solovyev, V.V. (2000). Ab initio gene finding in Drosophila genomic DNA. Genome Res 10, 516-22.
• Schatz et al. (1989). The V (D) J recombination activating genes, RAG -1. Cell 59, 1035-1048.
• Schlissel et al. (1993). Double-beach signal sequence breaks in V (D) J recombination are blunt, 5'-phosphorylated, RAG-dependent, and cell cycle regulated. Genes Dev 7, 2520-32.
• Schwarz et al. (1996). RAG mutations in human B cell-negative SCID. Science 274, 97-99.
• Shin et al. (1997). A Kinase-negative Mutation of Dna-Pkcs In Equine Scid Results In Defective Coding And Signal Joint Formation. J. Immunol. 158, 3565-3569.
• Shinkai et al. (1992). RAG-2 deficient mice varnish mature lymphocytes owing to inability to initiate V (D) J rearrangement. Cell 68, 855-867.
• Taccioli et al. (1998). Targeted disruption of the catalytic converter subunit of the DNA-PK gene in mice confers severe combined immunodeficiency and radiosensitivity. Immunity 9, 355-66.
• Taccioli et al. (1993). Impairement of V (D) J recombination in double-strand break repair mutants. Science 260, 207-210.
• Teo, S.H. and Jackson, S.P. (1997). Identification Of Saccharomyces Cerevisiae DNA ligase .4. Involvement In DNA Double-Beach Break Repair. EMBO Journal 16, 4788-4795.
• Tonegawa, S. (1983). Somatic generation of antibody diversity. Nature 302, 575-81.
• Van Gent et al. (1995). Initiation of V (D) J recombination in a cell-free system. Cell 87, 925-934.
• van Gent et al. (1996). Similarities between initiation of V (D) J recombination and retroviral integration. Science 271, 1592-1594.
• Varon et al. (1998). Nibrin, a novel DNA double-strand break Repair protein is mutated in Nijmegen breakage syndrome. Cell 93, 467-76.
• Villa et al. (2001). V (D) J recombination defects in lymphocytes due to RAG mutations: severe immunodeficiency with a spectrum of clinical presentations. Blood 97, 81-88.
• Wilson et al. (1997). Yeast DNA ligase IV mediates non-homologous DNA end-joining. Nature 388, 495-8.
• Zhu et al. (1996). Ku86 deficient mice exhibit severe combined Immunodeficiency and defective processing of V (D) J recombination intermediates. Cell 86, 379-389.
• Zhu, C. and Roth, D.B. (1995). Characterization of coding ends in thymocytes of scid mice: implications for the mechanism of V (D) J recombination. Immunity 2, 101-12.

SEQUENCE LISTING

Claims (30)

Isolated nucleic acid molecule that is selected from the group consisting of SEQ ID NO: 1, nucleotides 39-2114 of SEQ ID NO: 1 or nucleotides 60-2114 of SEQ ID NO: 1. Isolated nucleic acid molecule that is selected from the group consisting of nucleotides 39-1193 or the nucleotides 60-1193 of SEQ ID NO: 1. Isolated nucleic acid molecule containing the complement of the isolated nucleic acid molecule of claim 1 is. A vector comprising the nucleic acid molecule of claim 1. A host cell comprising the vector of claim 4. Method of producing a protein that participates in V (D) J recombination and / or DNA repair, comprising the steps of: a) Expressing the nucleic acid molecule of claim 1 in a suitable host to synthesize a protein that involved in V (D) J recombination and / or DNA repair, and b) Isolate the protein following V (D) J recombination and / or DNA repair is involved. Isolated protein or peptide produced by the nucleic acid of Claim 1 is encoded. Monoclonal or polyclonal antibody, the specifically recognizes the protein or peptide of claim 7. In vitro method for determining the type of severe combined immunodeficiencies (SCID) in a patient in it exists, the nucleic acid to analyze the selected is selected from the group consisting of SEQ ID NO: 1, nucleotides 39-2114, 60-2114, 39-1193 and 60-1193 of SEQ ID NO: 1 of the patient, wherein a mutation in the nucleic acid the Classification of the SCID as a radiation-sensitive SCID allows. In vitro diagnostic procedure, including a prenatal Diagnosis, a condition in a patient which is selected from the group consisting of a SCID, a predisposition for cancer, one Immunodeficiency and carrying a mutation, which increases the risk that Offspring have such a disease, consisting in the Analysis of the nucleic acid, the selected is selected from the group consisting of SEQ ID NO: 1, nucleotides 39-2114, 60-2114, 39-1193 and 60-1193 of SEQ ID NO: 1 and / or the protein bound by the nucleic acid of the Patients is encoded, with a mutation in the nucleic acid and / or the protein an elevated Risk for indicates the presence of a SCID. Method for identifying a connection that to the nucleic acid of claim 1 or 3 or can bind the protein of claim 7, comprising the steps: a) contacting the nucleic acid or of the protein with a candidate compound and b) Determine the binding between the candidate compound and the nucleic acid or the protein. One of the nucleic acids of claim 1 or 7, the vector of claim 4, the host cell of claim 5, the protein of claim 7 and the antibody of claim 8 as a medicament. A pharmaceutical composition comprising a pharmaceutically acceptable excipient with at least one of nucleic acid of claim 1 or 3, the vector of claim 4, the host cell of Claim 5, the protein of claim 7 and the antibody of Claim 8. Use of at least one of the nucleic acids of Claim 1 or 3, the vector of claim 4, the host cell of Claim 5, the protein of claim 7, the antibody of Claim 8 and the pharmaceutical composition of claim 13 for the preparation of a drug necessary for the therapy of a serious Combined immunodeficiency (SCID) is determined. Use of at least one of the nucleic acids of Claim 1 or 3, the vector of claim 4, the host cell of Claim 5, the protein of claim 7, the antibody of Claim 8 and the pharmaceutical composition of claim 13 for the preparation of a drug intended for the treatment of cancer is preferred, together with a genome-damaging agent, wherein the use is simultaneous, separate or sequential. Transgenic non-human mammal expressing the nucleic acid sequence of claim 1 integrated into its genome, which operationally linked to regulatory elements wherein the expression of the coding sequence is the concentration of the ARTEMIS protein represented by SEQ ID NO: 2 and / or the Measure of V (D) J recombination and / or DNA repair of the mammal relative to a non-transgenic mammal of the same species. Transgenic non-human mammal whose genome is a destruction of the endogenous ARTEMIS gene represented by SEQ ID NO: 1 being, being the destruction comprises the insertion of a selectable marker sequence and wherein the destruction The result is that the non-human mammal has a defect in the human mammal V (D) J recombination and / or DNA repair compared to a non-human wild-type mammal shows. Transgenic mammal according to claim 17, wherein the destruction is homozygous destruction. Transgenic mammal according to claim 18, wherein the homozygous destruction is a null mutation of the endogenous Gene encoding ARTEMIS represented by SEQ ID NO. 2 is shown. mammal according to claim 16 or 17, which is a mouse. Isolated nucleic acid comprising an ARTEMIS knockout construct, which comprises a selectable marker sequence, that of DNA sequences flanked homologous to the endogenous ARTEMIS gene, whose cDNA is represented by SEQ ID NO: 1, wherein when the construct into a non-human mammal or an ancestor of the non-human mammal is introduced in an embryonic stage, the selectable Marker sequence the endogenous ARTEMIS gene in the genome of the non-human mammal so destroyed, that the non-human mammal a defect in V (D) J recombination and / or DNA repair in Comparison to a non-human wild-type mammal shows. A vector comprising the nucleic acid of claim 21. Mammalian host cell, whose genome is a destruction of the endogenous ARTEMIS gene whose cDNA is identified by SEQ ID NO. 1, wherein the destruction is the insertion of a selectable Marker sequence includes. A method of screening compounds which V (D) J recombination and / or DNA repair, comprising contacting a The non-human mammalian compound of claim 17 or the host cell of claim 23 and determining the increase or decrease V (D) J recombination and / or DNA repair in the non-human mammal or the host cell compared to the V (D) J recombination and / or DNA repair of non-human mammal or the host cell prior to administration of the compound. Method for testing the genome toxicity of compounds, comprising contacting a compound with the non-human one mammal of claim 17 or the host cell of claim 23 and determining the increase or decrease in V (D) J recombination and / or DNA repair in the non-human mammal or the host cell compared to the V (D) J recombination and / or DNA repair of the non-human mammal or the host cell prior to administration of the compound. Use of at least one compound that selected is from the group consisting of the nucleic acid of claim 1 or 3, the vector of claim 4, the host cell of claim 5, the protein of Claim 7, the antibody of claim 8 and the pharmaceutical composition of claim 13 for the Preparation of a medicament for modulation of expression and / or activity of the ARTEMIS protein represented by SEQ ID NO: 2, in a cell. Non-human transgenic mammal whose genome is a first destruction, which is that of the endogenous ARTEMIS gene whose cDNA is represented by SEQ ID NO: 1, and comprises a second destruction, which is that of the endogenous p53 gene. Transgenic mammal according to claim 27, wherein at least the first destruction or the second destruction a homozygous destruction is. Transgenic mammal according to claim 27, in which the first destruction and the second destruction are homozygous destructions are. Transgenic mammal according to claim 27, which is a mouse.